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. 2020 Jul 14;11:3516. doi: 10.1038/s41467-020-17091-x

Fig. 5. IRSp53 directly binds RAB35 in its inactive GDP-bound state.

Fig. 5

a Lysates (1 mg) of HeLa cells transfected with GFP-RAB35 and Flag-IRSp53, serum starved overnight and treated (+) or not (−) with growth factors, were subjected to immunoprecipitation with anti-Flag (Flag) or control (Ctr) antibodies. Inputs (20 µg) and IPs were analyzed by immunoblotting with the indicated antibodies. pAKT was used for the positive control for growth factor stimulation. b Structure function analysis. Equal amounts (50 nM) of recombinant purified IRSp53 full-length WT or the indicated fragments and mutant were spotted onto nitrocellulose and incubated with recombinant purified GST-RAB35S22N (50 nM). After washing, nitrocellulose membranes were immunoblotted with an anti-GST antibody. c Indicated amounts of recombinant GST-RAB35S22N were spotted onto nitrocellulose and incubated with equal amounts (30 nM) of either recombinant purified IRSp53 WT or IRSp53 I-BAR* (K142E, K143E, K146E, K147E). After washing, nitrocellulose membranes were immunoblotted with an anti-IRSp53 antibody. BSA (100 nM) was used as the negative control. d Cell lysates (2 mg) from MDCK cells transfected with GFP empty vector, GFP-IRSp53 wild-type (WT), GFP-IRSp53 I-BAR*, and RFP-RAB35S22N were subjected to immunoprecipitation with anti-GFP (GFP) or control (Ctr) antibodies. Inputs and IPs were immunoblotted with the indicated antibodies. e Top: MDCK IRSp53-KD or IRSp53-KD cells were infected to express murine GFP-IRSp53 wild-type (WT), I-BAR*(K142E, K143E, K146E, K147E), CRIB* (I268N), SH3* (I403P) and PDZ* (V522G), and were seeded as single cells on a Matrigel layer and left to grow for 24/36 h. The cysts were fixed and stained with anti-β-catenin (green) or anti-PODXL (red) antibodies, and DAPI (blue), or processed for epifluorescence to visualize GFP-IRSp53 (green), and stained with an anti-PODXL antibody (red) and DAPI (blue). Asterisks, PODXL multi-foci. Scale bar, 10 µm. Bottom left: Quantification of multi-foci cysts. Data are means ± SD. Three/ four-cell stage cysts were analyzed, as at least 20 cysts/experiment in n = 5 (IRSp53-KD) and n = 2 (WT, I-BAR*, CRIB*, SH3*, PDZ*) independent experiments. P value, student’s t-test two-tailed. *p < 0.05; **p < 0.01. Source data are provided as a Source Data file. Bottom right: Immunoblotting to detect IRSp53, GFP-IRSp53 wild-type and mutants, and vinculin.