Fig. 3

BIN1 and CD2AP interact with RIN3. RIN3-flag was expressed and purified from HEK293t cells, then MS was used to identify and define RIN3 interactomes. a: A set of 380 RIN3-interacting proteins were detected, and a GO analysis was conducted. The RIN3 interactomes were divided into 20 categories with respect to different cellular function. b: A protein interaction network highlighting the RIN3-interactome that regulate endocytic/vesicular transport, based on 28 highly enriched proteins (PSM value ≥5). The circle size indicated the degree of enrichment. RIN3 was in green, LOAD risk factors were in blue. Interaction between RIN3 and CD2AP/BIN was analyzed by co-IP. HEK293 cells were transfected with indicated vectors. Cell lysates were incubated with either control IgG (IgG) and anti-flag IgG. Untransfected cells were used as a control. In c, the cell lysate inputs, the post-IP supernatant and the IP complexes were analyzed by SDS-PAGE/immunoblotting with indicated antibody. In d, RIN3-GFP was co-IP with CD2AP-flag; In e, RIN3-GFP was co-IP with BIN1-flag. For both D, E, a control IgG (IgG) was used as a negative control. RIN3-GFP was blotted with a GFP antibody, an anti-flag antibody was used to detect CD2AP-flag (d) or BIN1-flag (e). e Yeast two hybrid assay was performed, each group had three replicants. Standard t-test, **** stands for p < 0.0001, *** stands for p < 0.001, n.s. stands for p > 0.05