Figure 6.

Acetylation of TPD52 at lysine 163 impairs its binding to HSPA8 and inhibits the activation of CMA. (A) IB analysis of the whole-cell lysates (WCLs) and anti-FLAG IPs derived from the 293 T cells transfected with HA-KAT2B and the indicated mutation constructs of FLAG-TPD52. (B) IB analysis of the WCLs and anti-FLAG IPs derived from the 293 T cells transfected with GFP-HSPA8, FLAG-TPD52 wild type (WT), FLAG-TPD52^K163R and FLAG-TPD52^K163Q. (C) Identification of TPD52 acetylation sites (K163) using mass spectrometry (MS) analysis. The MS/MS spectrum of modified “KTSETLSQAGQKASAAFSSVGSVITK^{(Acetylation)}K” was shown. (D) IB analysis of the WCLs and anti-HA IPs derived from the 293 T cells transfected with HA-HSPA8, MYC-MEF2D, FLAG-TPD52 wild type (WT) and FLAG-TPD52^K163Q. (E) IB analysis of the WCLs derived from the PC3 cells transfected with FLAG-TPD52 wild type (WT) and FLAG-TPD52^K163Q. (F) Native continuous gel electrophoresis and IB analysis of lysosomes purified from the PC3 cells transfected with vector (Vec), FLAG-TPD52 wild type (WT) and FLAG-TPD52^K163Q