Fig. 4

TRIM45 regulates Atg5 based on its E3 ubiquitin ligase activity. a Western blot analysis of Atg5 expression in BV2 cells in response to different interventions. b BV2 cells with TRIM45 knockdown or control cells were treated with cycloheximide (CHX) for 0, 2 and 4 h. c BV2 cells were immunoprecipitated with IgG and anti-Atg5 antibodies, and the expression of TRIM45 and Atg5 was detected by western blotting. d Protein confidence analysis of Atg5 and TRIM45 was performed by high-throughput interactive group (TRIM45 (green), Atg5 (blue)). e BV2 cells were transfected with HA-TRIM45 for 48 h. BV2 cells were immunoprecipitated with an anti-Atg5 antibody and then subjected to western blot analysis with an anti-NLRP3 antibody. f Co-IP analysis of Atg5 ubiquitination in BV2 cells. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies. g HEK293T cells were transfected with TRIM45 and HA-tagged ubiquitin plasmids with or without LPS + ATP treatment. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies. h, i HEK293T cells were transfected with plasmids TRIM45 and HA-tagged k63 or k48 ubiquitin with or without LPS + ATP treatment. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies