Secondary antibodies coupled to horseradish peroxidase

Monoclonal rat anti-VANGL2 (2G4 monoclonal antibody) was generated as described21 (link). Anti-p62/SQSTM1 antibodies used in the study are as follows: mouse antibody (BD Biosciences 610833) or M01, clone 2C11, for EM experiments (Abnova: H00008878-M01) and guinea-pig polyclonal anti-p62 (Progen Biotechnik, GP62-C). Other antibodies used are as follows: rabbit antibody to LC3 (MBL Technology: PM036), rabbit antibody to GFP Abcam (ab290) and mouse antibody to α-tubulin (Sigma: B512), rabbit antibody to phospho-Thr183/Tyr185 SAPK/JNK (Cell Signalling: 9251) and SAPK/JNK (Cell Signalling: 9252), rabbit anti-β-catenin antibody (Santa Cruz Biotechnology: H102), mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (Abcam: ab9484), mouse monoclonal ubiquitin antibody (Life Sensors: FK2 AB120) and secondary antibodies coupled to horseradish peroxidase (Jackson Immunoresearch). Alexa Fluor-conjugated antibodies were purchased from Molecular Probes, Invitrogen. Antibodies were used according to the recommendations of the manufacturers or associated references.

The following antibodies were used: anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-phospho-Src family (Tyr416), anti-Yes, anti-Akt, anti-phospho-Akt (Ser473) and anti-p130Cas-pY410 from Cell Signalling Technology (Beverly, MA, USA); anti cortactin p80/85 (clone 4F11) and anti-phospho-cortactin (Tyr421) from Millipore (Temecula, CA USA); anti-β-catenin antibody (clone 6F9) and the monoclonal anti-phospho-ERK1/2 (Thr202/Tyr204) from Sigma-Aldrich (St. Louis, MO, USA); anti-E-cadherin and anti-p130Cas from BD Biosciences (Le Pont de Claix, France); anti-ERK1 (C-16) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Src and anti-Yes from R&D Systems (Abingdon, UK). Secondary antibodies coupled to horseradish peroxidase were from Jackson Immunoresearch Labs (West Grove, PA, USA). Fluorescently-labeled secondary antibodies were purchased from Invitrogen (Cergy Pointoise, France).

proteins were extracted using a Triton based buffer (0.5 % Triton, 300 mM Sucrose, 100 mM NaCl, 10 mM PIPES, 3 mM MgCl₂*6H₂O, 5 mM EDTA) supplemented with 1 μM DTT, 1 μM PMSF, 1 μM Pepstatin, 1 μg/ml Aprotenin, 0.5 μg/ml Leupeptin and phosphatase inhibitor cocktail 2 (Sigma, Cat. No. P5726). Following 10 min on ice the lysate was centrifuged at 14,000 rpm and the pellet discarded. Antibodies used for detection in western blot are as follows: Anti-phospho EGFR Y1092 (Abcam, 1:1000 Cat. No. ab40815), Anti-EGFR (Abcam, 1:1000 Cat. No. ab2430), anti-phospho AKT S473 (Cell signaling, 1:1000, Cat. No. 4058), Anti-AKT (Cell signaling, 1:1000, Cat. No. 11E7), anti-paxillin (Santa Cruz, 1:200, Cat. No. sc-136297), anti-beta-Actin (Abcam, 1:1000,Cat. No. ab6276), Strepavidin coupled HRP (Jackson Immunoresearch Laboratories, 1:1000). Secondary antibodies coupled to horseradish peroxidase (Jackson Immunoresearch Laboratories).