Secondary horseradish peroxidase antibodies

To measure APC levels in cells after silencing and rescue, cells were collected 48 h after initial oligo transfection and incubated for 30 min at 4°C in lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.5, 2 mM EDTA, 0.2 mM sodium orthovanadate, 20 mM β-glycerolphosphate, 50 mM sodium fluoride, 1 mM PMSF, 1 mM DTT, and 1× Roche complete protease inhibitor mixture). Samples were precleared by centrifugation at 14,000 rpm for 30 min at 4°C, quantified by Bradford assay, and immunoblotted. For measuring phospho-Paxillin, total Paxillin, and phospho–FAK-pY397 levels, the same procedure was used except that cells were incubated in serum-free media overnight before collection. Blots were probed with 1:500 rabbit anti-APC (ab15270; Abcam), 1:2,000 rabbit anti–GFP (ab6556; Abcam), 1:500 rabbit anti–phospho-Paxillin (Tyr118; PP4501; ECM Biosciences), 1:1,000 rabbit anti–FAK-phospho–tyrosine 397 (FAK-pY397; 141–9; Invitrogen), 1:1,000 mouse/human anti-Paxillin (AHO0492; Invitrogen), or 1:2,000 mouse anti–α-tubulin antibody (sc-32292; Santa Cruz Biotechnology). Blots were then probed with secondary horseradish peroxidase antibodies (GE Healthcare), and proteins were detected using a Pierce ECL Western Blotting Substrate detection kit (Thermo Fisher Scientific). Bands were quantified using Fiji.