Figure 3. S211 is a novel MTORC1-dependent phosphorylation site in TFEB required for the binding to YWHA. (A) Coomassie blue stained SDS-PAGE gel of co-immunopreciptated TFEB and YWHA from HeLa (CF7) cells treated with either DMSO or PP242. The asterisk (*) indicates IgG heavy and light chains. The arrowhead denotes actin. (B) Immunoblotting analysis of immunoprecipitates obtained as in (A). (C) Immunoblotting analysis of immunoprecipitates obtained as in (A) and incubated with or without calf intestinal alkaline phosphatase (CIAP). (D) Multi-sequence alignment of human TFEB S211 phosphorylation site with MITF and TFEB orthologs. (E) Immunoblotting analysis of immunoprecipitates from ARPE-19 cells overexpressing amino acid specific mutants and truncated forms of TFEB. (F) Collision-induced dissociation (CID) spectrum obtained for the TFEB S211 containing peptide (VGVTSSpSCPADL) showing phosphorylation at Serine 211. (G) Extracted ion chromatograms (XIC) of the peptide (m/z = 636.766) from cells treated with either DMSO or PP242.