Extended Data Fig. 6: Synthetic SPP-GFP interacts with endogenous proteins of HEK293 cells.
a, Western blot of wild-type HEK293 cells and HEK293 cells transfected with mRFP-Q74. Anti-mCherry and anti-polyQ-expanded antibodies were used to detect soluble mRFP-Q74. Both anti-mCherry and anti-polyQ-expanded antibodies detected two common bands of soluble mRFP-Q74 with different electrophoretic mobilities, that is the most intense band of ~55 kDa and another band of ~43kD. β-actin is the loading control. Representative of 9 independent experiments. b, Volcano plot of the interactome of synthetic SPP-GFP in HEK293 cells. Graph represents the –log10 (P-value) of a two-tailed t-test plotted against the log2 fold change of protein label-free quantification (LFQ) values from co-immunoprecipitation experiments using anti-GFP antibody. HEK293 cells expressing SPP-GFP were compared with HEK293 cells expressing control GFP. Red dots indicate significant interactors of SPP-GFP when compared to control GFP after correction for multiple testing. Student’s t-test (n = 3 biological replicates), False Discovery Rate (FDR)-adjusted P value (q value) <0.05 was considered significant. c, Chymotrypsin-like proteasome activity in HEK293 cells (relative slope to control GFP HEK293 cells). Graph represents the mean ± s.e.m. of three independent experiments. All the statistical comparisons were made by two-tailed Student’s t-test for unpaired samples. d, Western blot of HEK293 cells with anti-LC3 antibody to monitor autophagy flux. LC3-I is conjugated to phosphatidylethanolamine to form LC3-II, which amounts reflect the number of autophagosomes and autophagy-related structures. As a control, we treated wild-type HEK293 cells with 250 nM bafilomycin A (8 h), an inhibitor of autophagy that greatly increases the amount of LC3-II. β-actin is the loading control. Representative of three independent experiments.