Figure 3. Identification of RIM-BP2 as an exophilin-8-interaction protein.
(A) MIN6 cells expressing MEF-exophilin-8 or control LacZ protein were lysed and subjected to MEF-tag based purification. Bound proteins were detected by SDS-PAGE and Oriole fluorescent gel staining. Six bands specific to MEF-exophilin-8 are numbered. The number 1 band was identified as RIM-BP2 by LC-MS/MS analysis. (B) MIN6 cells were infected by adenovirus encoding MEF-exophilin-8 or control GFP protein. The immunoprecipitates (IP) with anti-FLAG antibody, as well as 1/100 of the original extracts, were immunoblotted with anti-FLAG and anti-RIM-BP2 antibodies. (C) The immunoprecipitates with anti-exophilin-8 antibody or control immunoglobulin G (IgG) in INS-1 832/13 cells, as well as 1/100 of the original extract, were immunoblotted with anti-exophilin-8 and anti-RIM-BP2 antibodies. (D) The total islet protein lysates (20 μg) from wild-type (WT) or exophilin-8-knockout (KO) mice were analyzed by immunoblotting with antibodies against exophilin-8, RIM-BP2, and α-tubulin. (E) The protein lysates (20 μg) from wild-type mouse tissues or cultured β-cell lines were analyzed by immunoblotting with anti-RIM-BP2 and anti-α-tubulin antibodies.