Fig. 4
DRD1 signaling promotes RIPK1 ubiquitination and degradation via Chip. A Primary neurons were subjected to SI and then treated with A-68930 (20 μM) for 1 h . Immunoblot analysis of K48-Ub in cell lysates immunoprecipitated with RIPK1 antibody (n=5/group). B List of E3 ligase proteins for top peptide hits identified in mass spectrometry study. (n=5/group). C Immunoblot analysis of RIPK1 and b-actin in cell lysates from A-68930-treated neurons transfected with siRNA against Chip (n=5/group). D Neurons were transfected with siRNA against Chip. After 48 h, the cells were subjected to SI and then treated with A-68930 (20 μM) for 1 h. Immunoblot analysis of K48-Ub protein from the cell lysates immunoprecipitated with anti-RIPK1 antibody (n=5/group). E Neurons were subjected to SI and then treated with A-68930 (20 μM) for 1 h. Immunoblot analysis of RIPK1 protein from the cell lysates immunoprecipitated with anti-Chip antibody (n=5/group). F Neurons were transfected with plasmids expressing HA–Ub, His–RIPK1 and Flag–Chip and then treated with 3-MA. The cell lysates were immunoprecipitated using anti-His antibody. RIPK1 ubiquitylation was detected by WB using anti-HA antibody (n=5/group). G Neurons were transfected with plasmids expressing with or without Flag–Chip or the Flag–Chip H260Q mutant. Immunoblot analysis of HA and RIPK1 from the cell lysates immunoprecipitated with anti-His antibody (n=5/group). H Beclin1-/+ mice were subjected to CCI. Immunoblot analysis of K48-Ub from the cortex lysates immunoprecipitated with anti-RIPK1 antibody (n=5/group). I Beclin1-/+ mice were subjected to CCI. Immunoblot analysis of RIPK1 from the cortex lysates immunoprecipitated with anti-Chip antibody (n=5/group)