Figure 5.

SQSTM1 mediates the lysosomal degradation of SCAP induced by lycorine. (A) A scheme for identified the proteins interacted with SCAP, which promoted by lycorine using IP-MS. (B) The experimental results were presented by volcano maps. (C) The top ten proteins interacting with SCAP promoted by lycorine. (D) 293 T cells were transfected with or without the indicated plasmids for 24 h. Immunoblotting for indicated proteins after immunoprecipitation of MYC from 293 T cells (n = 3). (E) HL-7702 cells were treated with or without 20 µM lycorine for 8 h. Immunoblotting for indicated proteins after immunoprecipitation of SCAP from HL-7702 cells (n = 3). (F) The schematic diagram of SCAP and its truncations. (G) 293 T cells transfected with Flag-SQSTM1 and the indicated HA-tagged SCAP constructs treated with 20 µM lycorine for 8 h and were subjected to immunoprecipitation with an anti-Flag antibody and levels of the co-immunoprecipitated HA-SCAP were detected with an anti-HA antibody (n = 3). (H) The schematic diagram of SQSTM1 and its truncations. (I-J) 293 T cells transfected with MYC-tagged SCAP and indicated Flag-SQSTM1-truncated constructs were treated with 20 µM lycorine and then were subjected to immunoprecipitation with an anti-MYC antibody. The levels of the co-immunoprecipitated Flag-SQSTM1 truncations were detected with an anti-Flag antibody (n = 3). (K) 293 T cells transfected with MYC-tagged SCAP and Flag-tagged SQSTM1TB (TRAF6 binding) domain (170–260) constructs were treated with 20 µM lycorine and then were subjected to immunoprecipitation with an anti-MYC antibody. The levels of the co-immunoprecipitated Flag-SQSTM1 TB domain was detected with an anti-Flag antibody (n = 3). Error bars are represented as mean ± SEM. Statistical analysis was done with one-way ANOVA (Dunnett’s posttest). *p < 0.05, **p < 0.01, ***p < 0.001