Figure 4.

Interaction of TPD52 with HSPA8 facilitates the activation of CMA. (A) Representative image and fluorescence photomicrographs of TPD52 and HSPA8 in C4-2 cells obtained via confocal microscopy. Scale bars represent 10 μm. (B) IB analysis of the whole-cell lysates (WCLs) and anti-FLAG IPs derived from the 293 T cells transfected with FLAG-TPD52 and HA-HSPA8. (C) IB analysis of the WCLs and anti-HSPA8 IPs derived from C4-2 cells. Immunoglobulin G (IgG) served as a negative control. (D) GST affinity-isolation assay revealed the direct interaction between TPD52 and HSPA8. The upper panel presents the IB results using the antibody against HIS, and the lower Coomassie blue stained gels show the purified proteins. (E) IB analysis of the WCLs and anti-HSPA8 IPs derived from TPD52-knockdown (shTPD52) and scramble (Sc) C4-2 cells. Immunoglobulin G (IgG) served as a negative control. (F) A schematic of the TPD52 structural domains used for mapping the sites of HSPA8 interaction. (G) IB analysis of the WCLs and anti-HA IPs derived from the 293 T cells transfected with GFP-HSPA8 and the indicated constructs of HA-TPD52. (H) IB analysis of the WCLs and anti-HA IPs derived from the 293 T cells transfected with GFP-HSPA8 and the indicated constructs of HA-TPD52. (I) IB analysis of the WCLs and anti-MYC IPs derived from 293 T cells transfected with GFP-HSPA8, MYC-MEF2D and the indicated constructs of HA-TPD52. (J) IB analysis of the WCLs derived from the PC3 cells transfected with the indicated constructs of HA-TPD52. (K) IB analysis of lysosomal LAMP2A purified from the PC3 cells transfected with the indicated constructs of HA-TPD52. (L) A schematic for the HSPA8 structural domains used for mapping the sites of interaction with TPD52. (M) IB analysis of the WCLs and anti-FLAG IPs derived from the 293 T cells transfected with FLAG-TPD52 and the indicated constructs of HA-HSPA8. (N) IB analysis of the WCLs derived from the 293 T cells transfected with MYC-MEF2D, FLAG-TPD52 and the indicated constructs of HA-HSPA8