Figure 6.

The small-molecule RL71 promotes SERCA2–LC3B interaction. (A) Co-IP of SERCA2 with endogenous LC3B in SUM1315 cells 6 h after the treatment of 2 μmol/L RL71. (B) Co-IP of FLAG-SERCA2 with endogenous LC3B and (C) Co-IP of EGFP–LC3 with endogenous SERCA2 in the transfected SUM1315 cells 6 h after the treatment of 2 μmol/L RL71. (D) Western blot analysis of p62 and LC3B in SUM1315 cells, which were transfected with the indicated plasmids and then treated with 2 μmol/L RL71 for 0–12 h. (E) Co-IP of FLAG-SERCA2 or its mutant with endogenous LC3B in SUM1315 cells, which were transfected with the indicated plasmids and then treated with 2 μmol/L RL71 for 6 h. (F) Ribbon representation of SERCA2 in the LC3-bound E1 form and that in E2 form bound with both LC3 and RL71. The cytoplasmic part of SERCA2 consists of N domain (yellow), P domain (blue) and A domain (purple)³⁰. LIR (red) in P domain interacts with LC3 (green). RL71 (dark purple) binds to SERCA2 at the cleft on the luminal side of the ER¹⁴. TM, transmembrane. (G) Cartoon representation of SERCA2 in E1 or E2 form bound with LC3 (gray). Red indicates high flexibility and green indicates low flexibility. Data are shown as the mean ± SD of at least three independent experiments. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.001.