I don't see any justification for this: "These bands were identified as full-length β4- (β4-FL) and α6-subunits, respectively (Fig. 1B). The additional <150 kD fragment represented a truncated form of β4-integrin (β4-C) that was lacking most of its extracellular domain". How do authors know that? Why Coomassie staining looks so different than autoradiography? Where is 250KDa band?
"β4-C did not immunoprecipitate with α6-integrin (Fig. 1A)". The bands in a6 IP are relatively faint and the one for β4-C in Fig.1b is even fainter. I am not sure that the conclusion is correct... Where is β4-C on IP with B4 (autoradiography)? It appears it is not detected, so why Author would expect to see it in precipitates from a6?
"Strikingly, both β4-FL and particularly the β4-C could be detected at the surface of α6KO cells (Fig. 1D & F)". How authors can be sure that the 150KDa band (prominent) is specific to β4-C? It could be unspecific. So the conclusion is not legitimate.
"While clones from the α6-KO construct 2 (α6KO2) expressed significantly lower levels of β4-FL at the cell surface compared to α6KO1, all of them retained normal surface levels of β4-C and α6-KO clone 1 (α6KO1) displayed high levels of surface expressed β4-FL. Moreover, α6 was abundantly expressed at the surface of both Itgβ4-KO cell clones (Fig. 1E & G)." This experiment (and conclusions) is missing critical controls: (1) specificity for β4-C (as above) and control for surface biotinylation efficiency α6KO2 (IP of Ecad).
Fig. 1 H-K. Surface immunoreactivity changes in KO cells. It would be helpful to add controls for antibodies - staining for Itg6 in intg6-KO and staining for Itg4 in Itg4-KO (TIRF mode basal membrane). Authors should also comment why surface expression of Itg4 is different in α6KO1 and α6KO2.
Authors should indicate in the text that Fig. 1M is confocal mode and not TIRF (like Fig. 1H-I), it would help to understand the data. Why such change in imaging mode was made is not explained in the text. Authors should analyze single and double KO in the same imaging mode or provide additional images for 2xKO in TIRF mode.
"Our data showed that β4-integrin is able to reach the cell surface in the absence α6-integrin" - "of" is missing.
"β4-integrin exists at the cell surface also in the absence of α6-subunit expression, suggesting that it can mature as a single subunit" - it is not clear why Authors comment on "maturation". Do they mean processing in the secretory pathways? Cell surface delivery? Please explain or be more precise.
I am not sure if I am interpreting the results correctly, but Supp. Fig. 2 A shows lack of Itg4 staining on the plasma membrane in the absence of Itg.6. Any comments on that?
In general, I have concerns regarding the experiments and some conclusions. The description in the text is often not precise making the manuscript difficult to read and follow the logic.