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Discipline
Biological
Keywords
LL5b
Adhesion
Exocytosis
Actin
Muscle
Observation Type
Standalone
Nature
Standard Data
Submitted
Feb 8th, 2016
Published
Nov 17th, 2016
  • Abstract

    LL5β is a peripheral membrane protein involved in cytoskeleton organisation, regulation of exocytosis and cellular adhesion. Protein-complex purification for TAP-tagged LL5β combined with mass spectrometry analysis identified an uncharacterised protein 2410002F23Rik as a potential LL5β-binding partner in muscle cells. Here we provide evidence that overexpressed 2410002F23Rik co-immunoprecipitates with LL5β in HEK293 cell lysate and that both proteins co-localise when expressed in HeLa. Collectively our results suggest that LL5β interacts with 2410002F23Rik, which we now name LL5β-interacting protein (LL5BIP).

  • Figure
  • Introduction

    Organisation of cellular architecture and plasma membrane turnover are fundamental aspects of cell function. LL5β plays an important role in the organisation of both microtubule and actin cytoskeleton, cellular adhesion, as well as regulation of sites at the cell cortex for fusion of exocytic vesicles with the plasma membrane. In skeletal muscles, LL5β was independently identified as a protein for which mRNA transcript is enriched at the neuromuscular junction, and which regulates the organisation of the postsynaptic machinery. Subsequent studies demonstrated that LL5β regulates exocytosis of postsynaptic acetylcholine receptors at the postsynaptic membrane. To perform its multiple cellular functions, LL5β recruits a number of downstream effectors, including a Rab6-binding protein ELKS. A recent biochemical screen for LL5β-binding partners identified several novel muscle-specific interacting proteins. Here we report that LL5β interacts with an uncharacterised protein 2410002F23Rik that we named LL5β-interacting protein (LL5BIP).

  • Objective

    To demonstrate the interaction between LL5β and 2410002F23Rik (LL5BIP).

  • Results & Discussion

    To identify proteins that interact with LL5β, TAP-tagged LL5β was expressed in differentiated C2C12 myotubes using adenovirus infection. Protein complexes were precipitated using IgG-coupled beads and eluted from the beads using TEV protease. Precipitates from lysates from uninfected cells served as a control. We performed a detailed analysis of the list of proteins that were detected by mass spectrometry (MS) as co-precipitating with LL5β. We discovered that one of the proteins that was recovered specifically with LL5β-binding beads but not in the control sample was the uncharacterised protein 2410002F23Rik (GenBank ID AAH16099; Fig. 1A). MS/MS analysis identified 15 peptides corresponding to 2410002F23Rik in the sample from TAP-LL5β-expressing cells, while no peptides were detected in the control purification. We compared the MS coverage of 2410002F23Rik in the precipitated sample to that of a known LL5β-binding protein ELKS, for which 56 peptides were detected in LL5β precipitates and 1 peptide in the control sample (Fig. 1A). The coverage of the protein sequences by the identified peptides was 31.1% for 2410002F23Rik and 36.1% for ELKS, suggesting that 2410002F23Rik is a high-confidence potential novel LL5β-binding partner. We therefore named it LL5β-interacting protein (LL5BIP).

    To verify the interaction between LL5β and LL5BIP we co-transfected HEK293 cells with GFP-LL5β and FLAG-LL5BIP constructs and performed co-immunoprecipitation from the cell lysate using anti-GFP antibody. Both proteins were expressed in HEK293 cells and detected in the lysate (input). FLAG-LL5BIP protein co-precipitated specifically with LL5β but did not precipitate on anti-GFP beads when expressed alone (Fig. 1B). Thus, LL5BIP interacts with LL5β in differentiated C2C12 myotubes and also in HEK293 cells upon overexpression. In agreement with these results both proteins showed significant level of co-localisation when overexpressed in HeLa cells (Fig. 1C).

  • Conclusions

    Using two biochemical approaches we have detected and verified the interaction between LL5β and an uncharacterised protein 2410002F23Rik, which we named LL5β-interacting protein (LL5BIP). In the first experiment overexpressed TAP-tagged LL5β interacted with endogenous LL5BIP and in the second experiment overexpressed proteins were co-immunoprecipitated from the HEK293 cell lysate. Further support to the notion that LL5β indeed interacts with LL5BIP comes from the microscopic observation that both proteins co-clocalise when overexpressed in HeLa cells.

  • Limitations

    Currently there are no antibodies that recognise endogenous 2410002F23Rik (LL5BIP), so we were not able to confirm interaction between endogenous LL5β and LL5BIP.

  • Methods

    Virus production and infection

    Adenoviral particles were produced by Welgen, Inc. (Worcester, MA, USA) from a vector encoding TAP–LL5β. C2C12 myotubes were infected with adenovirus on the second day after fusion induction and collected 2 days later. Lentiviral and retroviral particles were obtained from HEK-293 and GP2 cells respectively. Packaging cells were transiently transfected with plasmids. Media were replaced 24 h later, then collected 24 or 48 h later, centrifuged at 6000 rpm for 10 min and passed through a 0.45 µm filter. For infection, virus-containing medium was mixed with fresh medium containing Polybrene (Santa Cruz, CA, USA; SC-134220; 8 µg/ml).

    Complex purification and mass spectrometry analysis

    For complex purification, myotubes infected with TAP-GFP-LL5β adenovirus, or control (uninfected) myotubes, were washed with ice cold PBS containing sodium azide and covered with lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, 0.1% Nonidet-P40, 10% glycerol, 10 mM β-mercaptoethanol, EDTA-free Mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA), 1 mM phenylmethylsulphonyl fluoride; pH 8.0]. Cells were scraped off the dish, incubated briefly on ice, passed 3 times through a 25 gauge needle with syringe and centrifuged for 5 min at 4000 rpm and 30 min at 21000 rpm. The supernatant was incubated with washed IgG Sepharose 6 Fast Flow from GE Healthcare (Waukesha, WI, USA; 17-0969-01) for 4–16 h. Next, the Sepharose was loaded into a column (Bio-Rad, Hercules, CA, USA; 731-1550), washed 3 times with wash puffer (50 mM Tris-HCl, 50 mM NaH2PO4, 150 mM NaCl, 0.1% NP-40; pH 8.0), washed once with TEV buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT; pH 8.0) and resuspended in TEV buffer. To cleave the protein from the Sepharose, AcTEV (Invitrogen, Grand Island, NY, USA; 12575-015) was added and the sample was incubated for 2 h at room temperature or overnight at 4°C. For precipitation of proteins from eluted samples, 25% of the sample volume of 100% TCA (500 g TCA in 350 ml of H2O) was added, incubated for 10 min at 4°C and centrifuged at 14000 rpm for 5 min. The pellet was washed with 200 µl of cold acetone, centrifuged at 14000 rpm for 5 min, air dried and resuspended in sample buffer followed by incubation at 95°C for 6 min. For analysis, samples were subjected to SDS-PAGE electrophoresis and proteins were visualised with SilverQuest (Invitrogen, Grand Island, NY, USA; LC6070). Gels were stained with Colloidal Blue Staining Kit (Invitrogen, Grand Island, NY, USA; LC6025). Slices were excised from the gel and analysed at the Harvard Microchemistry and Proteomics Analysis Facility by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (μLC/MS/MS) on a Thermo LTQ-Orbitrap mass spectrometer.

    Cell cultures

    C2C12 cells were obtained from American Type Culture Collection (Manassas, VA, USA; CRL-1772). Cells were cultured for 5 or fewer passages in DME containing 20% foetal calf serum supplemented with glutamine, penicillin, streptomycin and Fungizone. Cells were trypsinised and replated onto 15 cm cell culture dishes. Before plating, dishes were coated with 10 µg/ml solution of laminin 111 (Invitrogen, Grand Island, NY, USA; 23017-015) in L-15 medium supplemented with 0.2% NaHCO3, incubated overnight at 37°C, and aspirated immediately before plating cells. To induce cell fusion, growth media was replaced with fusion media containing 2% horse serum in DMEM supplemented with glutamine, penicillin, streptomycin and Fungizone.

    HEK293 and HeLa cells were from American Type Culture Collection (Manassas, VA, USA; CRL-1573 and CCL-2, respectively) and were cultured in DMEM (Dulbecco's modified Eagle's medium, Lonza) containing 10 % FBS (Eurx), 1% glutamine and 1% penicillin/streptomycin (Life Technologies). Transfection of HEK293 and HeLa cells was performed using Lipofectamine 2000 (Invitrogen; 11668027) according to the manufacturer's instructions.

    Co-immunoprecipitation

    HEK-293 cells were transfected with appropriate constructs, washed with ice-cold PBS and covered with lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 0.1% Nonidet-P40, 10% glycerol, 1 mM DTT, EDTA-free Mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA); pH 8.0]. Cells were scraped off the dish, incubated briefly on ice, pipetted vigorously, and centrifuged for 30 min at 21000 rpm. Supernatants from centrifugations were incubated overnight with Dynabeads (Invitrogen, Grand Island, NY, USA; 658-01D) coated with anti-GFP antibody (Invitrogen, A-11120). Beads with attached proteins were washed four times with lysis buffer, resuspended in 2×SDS sample buffer and boiled for 5 min. For western blot analysis, samples were loaded on 10% acrylamide gels, subjected to the SDS-PAGE electrophoresis and transferred to the Biotrace NT nitrocellulose membrane (Pall; Port Washington, NY, USA). Anti-FLAG antibody (clone M2; Sigma, St. Louis, MO, USA; F1804-200UG) was used to detect FLAG-LL5BIP and the anti-GFP antibody used for western blot was from Epitomics (Burlingame, CA, USA; 1533-1).

    DNA cloning

    Mouse LL5BIP ORF (sequence ID BC016099) was amplified by PCR from C2C12 cDNA, previously generated from RNA isolated from C2C12 myotubes using High Capacity cDNA Reverse Transcription Kit (Life Technologies; Carlsbad, CA, USA). The primers used were: ATGGCTGAGAG (forward) and CTAGTCATGACGCAC (reverse) with appropriate overhangs for restriction enzyme digest. The PCR products were purified and cloned into FseI and AscI sites of pCDNA3.1/FFT-N/Puro, a custom-made plasmid based on the pCDNA3.1 backbone with the sequence of two tandem FLAG tags and a TEV restriction site upstream from the multiple cloning site (MCS) and FseI and AscI restriction sites engineered into the MCS. The resulting construct was FLAG-LL5BIP. Cloning of GFP-LL5β was described previously.

  • Funding statement

    This research was supported by Sonata-Bis 2012/05/E/NZ3/00487 grant from the National Science Center (NCN) in Poland. The project was carried out with the use of CePT infrastructure financed by the European Union- the European Regional Development Fund within the Operational Program ‘Innovative Economy’ for 2007-2013.

  • Ethics statement

    This work does not trigger ethical concerns.

  • References
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    Matters11.5/20

    LL5β interacts with uncharacterised protein 2410002F23Rik (LL5BIP)

    Affiliation listing not available.
    Abstractlink

    LL5β is a peripheral membrane protein involved in cytoskeleton organisation, regulation of exocytosis and cellular adhesion. Protein-complex purification for TAP-tagged LL5β combined with mass spectrometry analysis identified an uncharacterised protein 2410002F23Rik as a potential LL5β-binding partner in muscle cells. Here we provide evidence that overexpressed 2410002F23Rik co-immunoprecipitates with LL5β in HEK293 cell lysate and that both proteins co-localise when expressed in HeLa. Collectively our results suggest that LL5β interacts with 2410002F23Rik, which we now name LL5β-interacting protein (LL5BIP).

    Figurelink

    Figure 1. LL5β interacts with LL5BIP.

    (A) LL5BIP and ELKS were identified as LL5β interactors in a protein-complex purification experiment performed with differentiated C2C12 muscle cells. MS/MS spectra-total number of individual peptides identified by MS/MS; Protein coverage-% of protein sequence covered by identified peptides; Unique peptides-number of unique peptides identified by MS/MS; Mass (KDa)-protein mass in KDa; Protein length (aa)-protein length in amino acids. Peptides identified by MS/MS are indicated at the bottom.

    (B) GFP-LL5β co-immunoprecipitates with FLAG-LL5BIP when overexpressed in HEK293 cells. Results from two independent experiments are provided. Dark circles indicate constructs used for transfection of HEK293 cells; Input-cell lysate used for precipitation; IP:GFP-anti-GFP antibody immunoprecipitates.

    (C) LL5β co-localises with LL5BIP when expressed in HeLa cells.

    Introductionlink

    Organisation of cellular architecture and plasma membrane turnover are fundamental aspects of cell function. LL5β plays an important role in the organisation of both microtubule and actin cytoskeleton, cellular adhesion, as well as regulation of sites at the cell cortex for fusion of exocytic vesicles with the plasma membrane[1][2][3][4][5][6]. In skeletal muscles, LL5β was independently identified as a protein for which mRNA transcript is enriched at the neuromuscular junction, and which regulates the organisation of the postsynaptic machinery[7]. Subsequent studies demonstrated that LL5β regulates exocytosis of postsynaptic acetylcholine receptors at the postsynaptic membrane[8]. To perform its multiple cellular functions, LL5β recruits a number of downstream effectors, including a Rab6-binding protein ELKS[6]. A recent biochemical screen for LL5β-binding partners identified several novel muscle-specific interacting proteins[5]. Here we report that LL5β interacts with an uncharacterised protein 2410002F23Rik that we named LL5β-interacting protein (LL5BIP).

    Objectivelink

    To demonstrate the interaction between LL5β and 2410002F23Rik (LL5BIP).

    Results & Discussionlink

    To identify proteins that interact with LL5β, TAP-tagged LL5β was expressed in differentiated C2C12 myotubes using adenovirus infection. Protein complexes were precipitated using IgG-coupled beads and eluted from the beads using TEV protease. Precipitates from lysates from uninfected cells served as a control. We performed a detailed analysis of the list of proteins that were detected by mass spectrometry (MS) as co-precipitating with LL5β. We discovered that one of the proteins that was recovered specifically with LL5β-binding beads but not in the control sample was the uncharacterised protein 2410002F23Rik (GenBank ID AAH16099; Fig. 1A). MS/MS analysis identified 15 peptides corresponding to 2410002F23Rik in the sample from TAP-LL5β-expressing cells, while no peptides were detected in the control purification. We compared the MS coverage of 2410002F23Rik in the precipitated sample to that of a known LL5β-binding protein ELKS, for which 56 peptides were detected in LL5β precipitates and 1 peptide in the control sample (Fig. 1A). The coverage of the protein sequences by the identified peptides was 31.1% for 2410002F23Rik and 36.1% for ELKS, suggesting that 2410002F23Rik is a high-confidence potential novel LL5β-binding partner. We therefore named it LL5β-interacting protein (LL5BIP).

    To verify the interaction between LL5β and LL5BIP we co-transfected HEK293 cells with GFP-LL5β and FLAG-LL5BIP constructs and performed co-immunoprecipitation from the cell lysate using anti-GFP antibody. Both proteins were expressed in HEK293 cells and detected in the lysate (input). FLAG-LL5BIP protein co-precipitated specifically with LL5β but did not precipitate on anti-GFP beads when expressed alone (Fig. 1B). Thus, LL5BIP interacts with LL5β in differentiated C2C12 myotubes and also in HEK293 cells upon overexpression. In agreement with these results both proteins showed significant level of co-localisation when overexpressed in HeLa cells (Fig. 1C).

    Conclusionslink

    Using two biochemical approaches we have detected and verified the interaction between LL5β and an uncharacterised protein 2410002F23Rik, which we named LL5β-interacting protein (LL5BIP). In the first experiment overexpressed TAP-tagged LL5β interacted with endogenous LL5BIP and in the second experiment overexpressed proteins were co-immunoprecipitated from the HEK293 cell lysate. Further support to the notion that LL5β indeed interacts with LL5BIP comes from the microscopic observation that both proteins co-clocalise when overexpressed in HeLa cells.

    Limitationslink

    Currently there are no antibodies that recognise endogenous 2410002F23Rik (LL5BIP), so we were not able to confirm interaction between endogenous LL5β and LL5BIP.

    Methodslink

    Virus production and infection

    Adenoviral particles were produced by Welgen, Inc. (Worcester, MA, USA) from a vector encoding TAP–LL5β. C2C12 myotubes were infected with adenovirus on the second day after fusion induction and collected 2 days later. Lentiviral and retroviral particles were obtained from HEK-293 and GP2 cells respectively. Packaging cells were transiently transfected with plasmids. Media were replaced 24 h later, then collected 24 or 48 h later, centrifuged at 6000 rpm for 10 min and passed through a 0.45 µm filter. For infection, virus-containing medium was mixed with fresh medium containing Polybrene (Santa Cruz, CA, USA; SC-134220; 8 µg/ml).

    Complex purification and mass spectrometry analysis

    For complex purification, myotubes infected with TAP-GFP-LL5β adenovirus, or control (uninfected) myotubes, were washed with ice cold PBS containing sodium azide and covered with lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, 0.1% Nonidet-P40, 10% glycerol, 10 mM β-mercaptoethanol, EDTA-free Mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA), 1 mM phenylmethylsulphonyl fluoride; pH 8.0]. Cells were scraped off the dish, incubated briefly on ice, passed 3 times through a 25 gauge needle with syringe and centrifuged for 5 min at 4000 rpm and 30 min at 21000 rpm. The supernatant was incubated with washed IgG Sepharose 6 Fast Flow from GE Healthcare (Waukesha, WI, USA; 17-0969-01) for 4–16 h. Next, the Sepharose was loaded into a column (Bio-Rad, Hercules, CA, USA; 731-1550), washed 3 times with wash puffer (50 mM Tris-HCl, 50 mM NaH2PO4, 150 mM NaCl, 0.1% NP-40; pH 8.0), washed once with TEV buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT; pH 8.0) and resuspended in TEV buffer. To cleave the protein from the Sepharose, AcTEV (Invitrogen, Grand Island, NY, USA; 12575-015) was added and the sample was incubated for 2 h at room temperature or overnight at 4°C. For precipitation of proteins from eluted samples, 25% of the sample volume of 100% TCA (500 g TCA in 350 ml of H2O) was added, incubated for 10 min at 4°C and centrifuged at 14000 rpm for 5 min. The pellet was washed with 200 µl of cold acetone, centrifuged at 14000 rpm for 5 min, air dried and resuspended in sample buffer followed by incubation at 95°C for 6 min. For analysis, samples were subjected to SDS-PAGE electrophoresis and proteins were visualised with SilverQuest (Invitrogen, Grand Island, NY, USA; LC6070). Gels were stained with Colloidal Blue Staining Kit (Invitrogen, Grand Island, NY, USA; LC6025). Slices were excised from the gel and analysed at the Harvard Microchemistry and Proteomics Analysis Facility by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (μLC/MS/MS) on a Thermo LTQ-Orbitrap mass spectrometer.

    Cell cultures

    C2C12 cells were obtained from American Type Culture Collection (Manassas, VA, USA; CRL-1772). Cells were cultured for 5 or fewer passages in DME containing 20% foetal calf serum supplemented with glutamine, penicillin, streptomycin and Fungizone. Cells were trypsinised and replated onto 15 cm cell culture dishes. Before plating, dishes were coated with 10 µg/ml solution of laminin 111 (Invitrogen, Grand Island, NY, USA; 23017-015) in L-15 medium supplemented with 0.2% NaHCO3, incubated overnight at 37°C, and aspirated immediately before plating cells. To induce cell fusion, growth media was replaced with fusion media containing 2% horse serum in DMEM supplemented with glutamine, penicillin, streptomycin and Fungizone.

    HEK293 and HeLa cells were from American Type Culture Collection (Manassas, VA, USA; CRL-1573 and CCL-2, respectively) and were cultured in DMEM (Dulbecco's modified Eagle's medium, Lonza) containing 10 % FBS (Eurx), 1% glutamine and 1% penicillin/streptomycin (Life Technologies). Transfection of HEK293 and HeLa cells was performed using Lipofectamine 2000 (Invitrogen; 11668027) according to the manufacturer's instructions.

    Co-immunoprecipitation

    HEK-293 cells were transfected with appropriate constructs, washed with ice-cold PBS and covered with lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 0.1% Nonidet-P40, 10% glycerol, 1 mM DTT, EDTA-free Mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA); pH 8.0]. Cells were scraped off the dish, incubated briefly on ice, pipetted vigorously, and centrifuged for 30 min at 21000 rpm. Supernatants from centrifugations were incubated overnight with Dynabeads (Invitrogen, Grand Island, NY, USA; 658-01D) coated with anti-GFP antibody (Invitrogen, A-11120). Beads with attached proteins were washed four times with lysis buffer, resuspended in 2×SDS sample buffer and boiled for 5 min. For western blot analysis, samples were loaded on 10% acrylamide gels, subjected to the SDS-PAGE electrophoresis and transferred to the Biotrace NT nitrocellulose membrane (Pall; Port Washington, NY, USA). Anti-FLAG antibody (clone M2; Sigma, St. Louis, MO, USA; F1804-200UG) was used to detect FLAG-LL5BIP and the anti-GFP antibody used for western blot was from Epitomics (Burlingame, CA, USA; 1533-1).

    DNA cloning

    Mouse LL5BIP ORF (sequence ID BC016099) was amplified by PCR from C2C12 cDNA, previously generated from RNA isolated from C2C12 myotubes using High Capacity cDNA Reverse Transcription Kit (Life Technologies; Carlsbad, CA, USA). The primers used were: ATGGCTGAGAG (forward) and CTAGTCATGACGCAC (reverse) with appropriate overhangs for restriction enzyme digest. The PCR products were purified and cloned into FseI and AscI sites of pCDNA3.1/FFT-N/Puro, a custom-made plasmid based on the pCDNA3.1 backbone with the sequence of two tandem FLAG tags and a TEV restriction site upstream from the multiple cloning site (MCS) and FseI and AscI restriction sites engineered into the MCS. The resulting construct was FLAG-LL5BIP. Cloning of GFP-LL5β was described previously[7].

    Funding Statementlink

    This research was supported by Sonata-Bis 2012/05/E/NZ3/00487 grant from the National Science Center (NCN) in Poland. The project was carried out with the use of CePT infrastructure financed by the European Union- the European Regional Development Fund within the Operational Program ‘Innovative Economy’ for 2007-2013.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    This work does not trigger ethical concerns.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
    1. Samantha J. Stehbens, Matthew Paszek, Hayley Pemble,more_horiz, Torsten Wittmann
      CLASPs link focal-adhesion-associated microtubule capture to localized exocytosis and adhesion site turnover
      Nature Cell Biology, 16/2014, pages 561-573 DOI: 10.1038/ncb2975chrome_reader_mode
    2. V. Astro, S. Chiaretti, E. Magistrati,more_horiz, I. de Curtis
      Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration
      Journal of Cell Science, 127/2014, pages 3862-3876 DOI: 10.1242/jcs.155663chrome_reader_mode
    3. Gideon Lansbergen, Ilya Grigoriev, Yuko Mimori-Kiyosue,more_horiz, Anna Akhmanova
      CLASPs Attach Microtubule Plus Ends to the Cell Cortex through a Complex with LL5β
      Developmental Cell, 11/2006, pages 21-32 DOI: 10.1016/j.devcel.2006.05.012chrome_reader_mode
    4. Varuni Paranavitane, Len R. Stephens, Phillip T. Hawkins
      Structural determinants of LL5β subcellular localisation and association with filamin C
      Cellular Signalling, 19/2007, pages 817-824 DOI: 10.1016/j.cellsig.2006.10.007chrome_reader_mode
    5. T. J. Proszynski, J. R. Sanes
      Amotl2 interacts with LL5β, localizes to podosomes and regulates postsynaptic differentiation in muscle
      Journal of Cell Science, 126/2013, pages 2225-2235 DOI: 10.1242/jcs.121327chrome_reader_mode
    6. Ilya Grigoriev, Daniël Splinter, Nanda Keijzer,more_horiz, Anna Akhmanova
      Rab6 Regulates Transport and Targeting of Exocytotic Carriers
      Developmental Cell, 13/2007, pages 305-314 DOI: 10.1016/j.devcel.2007.06.010chrome_reader_mode
    7. Masashi Kishi, Terrance T. Kummer, Stephen J. Eglen, Joshua R. Sanes
      LL5β
      The Journal of Cell Biology, 169/2005, pages 355-366 DOI: 10.1083/jcb.200411012chrome_reader_mode
    8. S. Basu, S. Sladecek, I. Martinez de La Pena Y Valenzuela,more_horiz, H. R. Brenner
      CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles
      Molecular Biology of the Cell, 26/2015, pages 938-951 DOI: 10.1091/mbc.e14-06-1158chrome_reader_mode
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