Fig. 5.

Pcdhα is bound by ESCRT-0 in undifferentiated cells. (A) Confocal imaging of Pcdhα4-TAP and ESCRT-0. CAD cells stably expressing Pcdhα4-TAP were transfected with the ESCRT-0 subunit Hrs fused to GFP. After 0 or 72 h of differentiation, cells were fixed and stained with anti-HA (red or grayscale), anti-GFP (green or grayscale), and DAPI (blue). (B) Coimmunoprecipitation of Pcdhα4-TAP and endogenous Hrs. CAD cells stably transfected with an empty vector or Pcdhα4-TAP were differentiated for 0 h (UD) or 72 h (D). Pcdh was immunoprecipitated from cell lysates with anti-FLAG antibody, and Western blots were carried out for Hrs and FLAG. A nonspecific band comigrates with Pcdhα4-CTF1 following FLAG IP. (C) Coimmunoprecipitation of endogenous Hrs and Pcdhα4-TAP. Lysates of undifferentiated cells stably transfected with an empty vector or Pcdhα4-TAP were used for immunoprecipitation with a polyclonal antibody against Hrs or a control IgG. Western blots were then carried out against FLAG and Hrs. (D) Coimmunoprecipitation of α4ΔEC1-5-TAP and Hrs. α4ΔEC1-5-TAP was immunoprecipitated from lysates of undifferentiated or differentiated cells and a Western blot was performed against Hrs and FLAG.