Irdye 800cw goat anti rabbit immunoglobulin g igg

Blebbistatin (B05060), low-endotoxin BSA (A8806), PA (P9767), and Y27632 (Y0503) were from Sigma Aldrich (St. Louis, MO), while cis-PO/palmitoleic acid (10009871) was from Cayman Chemicals (Ann Arbor, MI). Antibodies used for immunoblotting were α-actinin (Millipore Sigma; A7811), β-actin (Cell Signaling; 4790), α-catenin (ThermoFisher; 13-9700), β-catenin (BD Biosciences; 610153), VE-cadherin (Santa Cruz; 9989), RhoA (Santa Cruz; c-418), IRDye 800CW goat anti-rabbit immunoglobulin G (IgG) (LI-COR; 9253211), and IRDye 680LT goat anti-mouse IgG (92668070). Antibodies and reagents used for immunofluorescence were α-catenin (ThermoFisher; 13-9700), β-catenin (BD Biosciences; 610153), DAPI (4’,6-Diamidino-2-phenylindole, dihydrochloride; ThermoFisher; D1306), GM130 (BD Biosciences; 610823), phospho-MLC (Cell Signaling; 3674), phalloidin (Alexa Fluor 555) (ThermoFisher; A34055), phospho-YAP S127 (Abcam; 76252), YAP (Cell Signaling 14074), VE-cadherin (Santa Cruz; 9989), goat anti-rabbit IgG secondary antibody (Alexa Fluor 488) (ThermoFisher; A11008), goat anti-mouse IgG secondary antibody (Alexa Fluor 647) (ThermoFisher; A21235). Dako fluorescence mounting medium (S3023) was from Aligent. Y227632 (Y0503) was from Millipore Sigma (Burlington, MA). The plasmid encoding GFP–β-actin was kindly provided by Sergio Grinstein (Hospital for Sick Children, Toronto, Ontario, Canada).

Snap-frozen tumors were digested with radioimmunoprecipitation assay lysis buffer containing phosphatase inhibitors (Sigma). Protein concentrations were measured with a Pierce BCA protein assay (23227, Thermo Scientific, MA, USA). 50 μg of protein per sample was separated on 12% SDS-PAGE and then electro-blotted onto nitrocellulose membranes using the iBlot Dry Blotting System (Thermo Scientific). Membranes were blocked with 3% bovine serum albumin in PBS with 0.02% Tween 20 (PBST) for 1 h and then probed with rabbit anti-survivin antibody (1:10,000 dilution; Ab 76424, Abcam) at 4°C overnight (16 h). Following three washes with PBST, the membrane was incubated with IRDye 800CW goat anti-rabbit immunoglobulin G (IgG) (1:5,000 dilution, 925-32211, LI-COR Biosciences, NE, USA) for 1 h. Expression of GAPDH was used as a loading control using a mouse anti-GAPDH antibody (1:10,000 dilution, MAB374, Sigma) and IRDye 680CW goat anti-mouse IgG secondary antibody (1:5,000 dilution, 926-32220, LI-COR Biosciences). The fluorescent signal was measured using the Odyssey imaging system (LI-COR Biosciences). The same protocol was used to measure survivin levels in cell lysates.