Fig. 8: Arl4A affects the association of VPS36 and ESCRT-III components.

a HeLa cells were transfected with the indicated VPS36 plasmids and GFP-CHMP2A for 24 h and stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (red) and DAPI (blue). Scale bar, 10 µm. Quantification of VPS36-Myc and GFP-CHMP2A colocalization was obtained from three biological repeats and shown as the colocalization coefficient by ZEN software. The results represent the mean ± SD and the p-value was assessed by a two-sided t-test (VPS36-WT = 62 cells, VPS36-A6 = 74 cells). b Comparison of VPS36-WT and VPS36-A6 binding ability toward ESCRT-III component CHMP2A in vivo. HeLa cells transfected with indicated plasmids were treated with 1 mM dithiobis(succinimidyl propionate) (DSP) for 2 h and quenched by Tris–HCl before cell lysis and immunoprecipitation with GFP-Trap. The bound proteins were analyzed by western blotting with anti-Myc or anti-GFP antibodies. The amounts of coimmunoprecipitated VPS36 were determined by densitometric quantification from four experiments as indicated. The results represent the mean ± SD and the p-value was assessed by a two-sided t-test. c Co-IP of VPS36 and CHMP2A in Arl4-knockdown HeLa cells. Cells transfected with the control or Arl4A siRNA as well as the indicated plasmids were treated with 1 mM DSP for 1 h and immunoprecipited with GFP-Trap. The bound proteins were analyzed by western blotting with indicated antibodies. The amounts of coimmunoprecipitated GFP-CHMP2A were determined by densitometric quantification and represent the mean ± SD of three independent experiments (the p-value was assessed by a two-sided t-test). d and e Effects of Arl4A overexpression on the modulation of the d CHMP2A-USP8/UBPY interaction and e CHMP2A–AMSH interaction in 293T cells. Lysates of cells transfected with the indicated plasmids were immunoprecipitated with HA beads and bound proteins were analyzed by western blotting with anti-HA, anti-mCherry, or anti-Arl4A antibodies. The amounts of coimmunoprecipitated CHMP2A were determined by densitometric quantification from d three and e four experiments as indicated. The results represent the mean ± SD and the p-value was assessed by a two-sided t-test. Source data are provided as a Source Data file.