a Strategy for identifying epsin 1 binding proteins. b Coomassie-stained gel of the epsin 1 IP immunocomplex from MAECs. Bands were characterized by mass spectrometry. HC heavy chain, LC light chain. c, d MAECs isolated from WT mice were treated with 100 μg/mL oxLDL or 500 μg/mL cholesterol crystal (Chol) or 50 μM 7-KC for 30 min and an immunoprecipitation (IP) was performed using the epsin 1 antibody. The immunocomplex supernatant was probed with antibodies to IP3R1 or epsin 1 by western blot analysis (c) and then quantified (d) (n = 6 independent experiments, asterisk P < 0.05 compared with control). e, f Reciprocal IP using the IP3R1 antibody in the same samples as described in c and d. A representative western blot (e) and quantification (f) (n = 6 independent experiments, asterisk P < 0.05 compared to control) of the IP are shown. g, h MAECs isolated from control mouse aortas (i.e., from ApoE−/− mice) were pre-treated with 5 μM MG132 for 4 h followed by treatment with 100 μg/mL oxLDL or 500 μg/mL Chol or 50 μM 7-KC for 36 h in the presence of vehicle (DMSO) or MG132. IP3R1 was measured by western blot (g) and quantified (h) (n = 5 independent repeats, asterisk P < 0.001 compared to control). All data were assessed using Student’s t-test and are presented as the mean ± SEM.