He et al. showed that GSAP silencing specifically inhibited γ-cleavage of APP. This study was recently refuted by Udayar and Rajendran showing that GSAP silencing did not specifically inhibit Aβ, but it also lowered the sAPPβ levels, which is a product of β-cleavage of APP and not γ-cleavage. To reproduce this finding, we silenced GSAP using a pool of four siRNAs and assayed for secreted Aβ and sAPPβ levels. As controls, we used siRNAs against APP, BACE1, Pen-2, and a scrambled medium GC oligo (MedGC). The measurements to monitor APP processing were performed using a multiplexed system to quantitatively measure both Aβ and sAPPβ levels from the sample. We observed that GSAP silencing led to a decrease in both Aβ and sAPPβ levels, in agreement with the previously published finding (Fig. 1A). To study how GSAP affected both Aβ and sAPPβ levels, we first examined APP processing by Western blotting with anti-APP C-terminus-specific antibody. Surprisingly, GSAP silencing dramatically decreased full-length APP levels (Fig. 1B) (see independent replicate data in supplementary information; Supl Fig. 1A). As a control, we used siRNAs against APP and found that it significantly reduced APP protein levels (Fig. 1B), as expected. To rule out that the reduction in APP was due to general cell toxicity or defective cell viability, we performed a number of controls. First, we analyzed the levels of a cellular protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and found these to be unchanged in GSAP-silenced cells (Fig. 1B; Supl Fig. 1A) in the same blot along with anti-APP antibody. Second, the levels of Nicastrin, another type 1 transmembrane protein, were not affected in GSAP-silenced cells (Supl Fig. 1B). Third, cell viability measurements (of 20 replicates) by Alamar Blue™ from GSAP-silenced and scrambled oligo-treated control cells did not show any difference demonstrating that GSAP silencing did not induce any cell toxicity (Supl Fig. 1C). However, silencing Kif11, a kinesin involved in cell division, severely induced cell death, serving as a positive control for the cell toxicity assay measured by Alamar Blue (Supl Fig. 1C). Fourth, general morphological characterization showed no distinguishable effect of GSAP silencing on cell shape, structure, and number (Supl Fig. 1D). Fifth, release of the cytosolic protein, lactate dehydrogenase (LDH), was monitored to detect any membrane leakages or subtle cellular membrane defects. GSAP silencing did not affect the release of LDH (Supl Fig. 1E). We confirmed the efficiency of GSAP silencing by RT-PCR (Supl Fig. 1F). Together, our results conclusively show that GSAP silencing reduced full-length APP levels and thus altered both β- and γ-cleavage and not specifically γ-cleavage of APP or induced any cytotoxicity. To independently validate the effect of GSAP on APP levels and also to rule out any epitope-specific effects due to the use of the C-terminal antibody of APP that we used for Western blotting, we checked the cellular levels of APP using immunofluorescence with another anti-APP antibody, 6E10, that recognises the N-terminus of the Aβ domain. Again, silencing of GSAP substantially reduced APP levels (Fig. 1C) demonstrating that indeed GSAP regulates APP levels. Consistent with the observation that GSAP silencing reduces full-length APP levels, GSAP silencing also significantly reduced the levels of sAPPα, which is produced by the non-amyloidogenic (α-secretase) pathway (Fig. 1D). This reduction was not due to cell death, as the viability of GSAP-silenced cells was not affected (Fig. 1D; Supl Fig. 1). To rule out any off-target effects, we silenced GSAP using four different siRNAs singly and analyzed its effect on APP levels. We observed that each of the four siRNAs reduced APP levels individually (Supl Fig. 2A and Supl Fig. 2B), thus demonstrating the specificity of the observed effect of GSAP silencing on APP levels. Thus, GSAP regulates full-length APP levels and not specifically γ-secretase activity. To further validate our findings, we used yet another approach by using the anti-cancer drug, imatinib. Imatinib inhibits Aβ production, which was concluded to occur via binding to GSAP by blocking its activity . Since our results indicate that GSAP does not specifically regulate the γ-secretase activity, we then asked whether imatinib could regulate γ-secretase activity in a GSAP-independent manner. Treatment of cells with imatinib indeed selectively decreased Aβ levels without affecting sAPPβ levels or cell viability, as has been previously shown by several laboratories(Fig. 1E; Supl Fig. 1G). However, while cells silenced with GSAP siRNA again showed a reduction in both Aβ and sAPPβ levels, treatment with imatinib (10 mM, same concentration used in He et al.) caused a further significant and specific decrease in Aβ levels, suggesting that imatinib regulates Aβ levels independently of GSAP. Recent studies on imatinib and Aβ also fail to see an effect, further casting doubt that GSAP is an imatinib target. We have tried to reproduce the results  using the cells, plasmids, experimental conditions, concentrations of inhibitors as identical as those employed by He et al.,  but we failed to reproduce many of their findings. Due to the unavailability of a working anti-GSAP antibodies, we were unable to look at the cleavage of endogenous GSAP. However, in large-set experiments, using RNAi against GSAP, we studied the functionality of the endogenous GSAP. The only one result that we can reproduce is the effect of GSAP on Aβ levels, which we discovered to be not due to the effect of GSAP on γ-secretase activity as claimed by He et al.,  but we have shown that this is rather due to GSAP’s effect on APP.