Anti phospho cofilin ser3

The following antibodies were used in this study: anti-Rac1 (clone 23A8) from Millipore, anti-Cdc42, anti-RhoA and anti-Ezrin from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-Rap1 antibody from Epitomics (Burlingame, CA, USA) and Cell Signaling Technology (Boston, MA, USA), anti-cofilin, anti-phospho-cofilin (Ser3) and anti-phospho-ERM from Cell Signaling Technology, anti- mouse CD11a (clone M17/4) and anti-CD4 from eBioscience (San Diego, CA, USA).

Proteins were isolated from cells using M-PER (Thermo Scientific, MA, USA) separated in Mini-PROTEAN TGX Gel (4–12%, Bio-Rad) and electrotransferred onto a PVDF membrane (Millipore). After blocking in Blocking One (Nacalai Tesque, Kyoto, Japan), the membranes were incubated for 1 h at room temperature with primary antibodies, which included anti-CD63 (purified mouse anti-human CD63, H5C6, 1:200, BD), anti-CD9 (ALB6, 1:200, Santa Cruz Biotechnology Inc.), anti-cytochrome C (purified mouse anti-cytochrome C, 7H8.2C12, 1:200, BD), anti-Claudin-5 (Z43.JK, 1:200, Invitrogen), anti-Occludin (ZMD.481, 1:200, Invitrogen), anti-ZO-1 (H-300, 1:100, Santa Cruz Biotechnology), anti-N-cadherin (3B9, 1:500, Invitrogen), anti-PDPK1 (#3062, 1:500, Cell Signaling), anti-GAPDH (6C5, 1:1000, Millipore), anti-Cofilin (D3F9, 1:1000, Cell Signaling), anti-Phospho-Cofilin (Ser3) (#3311, 1:500, Cell Signaling) and anti-N-SMase2 (H-195, 1:200, Santa Cruz Biotechnology Inc.). Secondary antibodies (HRP-linked anti-mouse IgG, NA931 or HRP-linked anti-rabbit IgG, NA934, GE Healthcare) were used at a dilution of 1:2,000. The membrane was then exposed to ImmunoStar LD (Wako, Osaka, Japan). Original scans of the cropped images in the figures are presented in Supplementary Figs 7–13.

The following antibodies were used in this study (with dilution factor for immunoblotting): anti-PKN2 (#2612, 1:1,000), anti-YAP (#14074, 1:1,000), anti-YAP/TAZ (#8418, 1:1,000), anti-phospho-YAP (Ser127) (#4911, 1:1,000), anti-phospho-EGFR (Tyr1068) (#3777, 1:1,000), anti-EGFR (#4267, 1:20,000), anti-phospho-AKT (Ser473) (#4058, 1:1,000), anti-AKT (#9272, 1:5,000), anti-phospho-ERK1/2 (Thr202/Tyr204) (#9101, 1:1,000), anti-ERK1/2 (#9102, 1:5,000), anti-ARIH2/TRIAD1 (#13689, 1:1,000), anti-BTAF1 (#2637, 1:1,000), anti-GNAQ (#14373, 1:1,000), anti-HSP90 (#4877, 1:5,000), anti-ALDOA (#8060, 1:5,000), anti-METAP2 (#12547, 1:1,000), anti-GAPDH (#2118, 1:5,000), anti-RHOA (#2117, 1:1,000), anti-Cofilin (#5175, 1:1,000), anti-phospho-Cofilin (Ser3) (#3313, 1:1,000), from Cell Signaling Technology; anti-α-Tubulin (T6074, 1:20,000) and anti-β-Actin (A1978, 1:20,000) from Sigma; anti-GNB2 (ab81272, 1:1,000), anti-RIC8A (ab97808, 1:1,000), anti-USP22 (ab195289, 1:1,000), anti-CUL5 (ab184177, 1:1,000), anti-RNF7 (ab181986, 1:1,000), anti-PDCD10 (ab180706, 1:1,000), from Abcam; anti-KCTD5 (#15553–1-AP, 1:1,000), anti-PSAT1 (#10501–1-AP, 1:5,000), from Proteintech; Goat Anti-Rabbit IgG Antibody, (H+L) HRP conjugate (#AP307P, 1:5,000), Goat Anti-Mouse IgG Antibody, (H+L) HRP conjugate (#AP308P, 1:5,000), from Millipore Sigma.