Fig 1: 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
Fig 2: RIN3 upregulation in APP/PS1 BFCNs induce enlarged early endosome. E18 BFCNs of WT (a) and APP/PS1 (b) were cultured and fixed at DIV4 for immunostaining with Rab5 antibody. Nuclei were stained with DAPI. Rab5 positive vesicles were measure by diameter (c) and size (d) using ImageJ (in blue bar graph). The mRNA level for Rab5 (e), RIN3 (f) and RIN2 (g) were quantitated using qPCR (in red bar graph). Two tail T-test was used. * p < 0.05 and ***p < 0.001. As in A and B, E18 BFCNs of WT (h) and APP/PS1 (i) were also co-stained for RIN3 and MAP2. The inset in I shows enlarged RIN3 puncta. Representative images are shown
Fig 3: CD2AP and BIN1are recruited to early endosome by RIN3. PC12M cells expressing GFP/CD2AP-flag (a, b), RIN3-GFP/CD2AP-flag (c, d), GFP/BIN1-flag (e, f), RIN3-GFP/BIN1-flag (e-h) were immune-stained for Rab5 with a specific antibody. Representative images are shown. Colocalization analysis was performed by imageJ by measuring fluorescence intensity alongside the drawn line. The three different colors peak at the same position If the three proteins were colocalized (b, d, f, h)
Fig 4: RIN3 and CD2AP inhibited transport of APP and BACE1 in primary cortical neurons. E18 mouse cortical neurons were cultured in microfluidic chambers and were co-transfected with the indicated expression vectors. Live imaging was performed as described in the Methods section. Representative images of axons and corresponding kymographs are shown in a-h. The percentiles of mobile versus stationary vesicles (stat) (i, k) and average velocities (g, l) for APP-mCherry and BACE1-mCherry in axons were quantitated. Data represent mean ± SEM of at least 3 independent experiments. All p-values were calculated using 1-way ANOVA. p < 0.05 (*), p < 0.01(**), p < 0.0001(****), p > 0.05 (n.s.), standard t-test
Fig 5: RIN3 promotes APP CTFs production and Tau phosphorylation via Rab5. PC12M cells were transfected with GFP, RIN3-GFP, BIN1-GFP, CD2AP-GFP and cell lysates were immunoblotted using indicated antibodies (a). In b, PC12 cells were transfected with indicated vector(s) (RIN3-mCherry, Rab5S34N-GFP). and cell lysates were analyzed by SDS-PAGE/immunoblotting with either the APP C-terminals antibodies to detect full length APP/APP CTFs and other antibodies as indicated. Full-length APP (fl APP), APP CTFs, total Tau and phosphorylated Tau are shown (a and b). Both the ratio of CTFs/APP-fl and the ratio of pTau/total Tau is shown in c and d. p < 0.05 (*), p > 0.05 (n.s.), standard t-test
Supplier Page from Abcam for Anti-RIN3 antibody