Hrp conjugated anti rabbit igg or anti mouse igg

Whole-cell protein was isolated as described previously[42 (link)]. For tissue protein extraction, the tumor tissues were homogenized in ice-cold RIPA (radioimmunoprecipitation assay) lysis buffer (Millipore). The tissue lysates were incubated at 4°C for 1 hour with rotation, followed by clarification of tissue debris by centrifugation at 12,000 rpm for 10 minutes. The protein concentration of tumor extracts was determined using the BCA Protein Assay Kit (Pierce). Western blotting were performed as previously described[42 (link)]. Antibody binding was revealed using an HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millopore) and exposure to X-Omat film (Kodak).

Cells were lysed with cell lysis buffer (0,5% NP-40, 150 mM NaCl, 50 mM Tris-HCl, 0,25 mM EDTA, 1 mM DTT, 1 mM Na₃VO₄ and 1:2000 protease inhibitor cocktail (all from Sigma-Aldrich)) as in [46 (link)]. 40 μg total proteins were resolved by SDS-PAGE and electroblotted onto PVDF membranes. After blocking with non-fat dry milk diluted in Tris-Buffered Saline – Tween (TTBS) 0,05%, membranes were probed with rabbit anti-SCARA5 (Santa Cruz Biotechnology) or mouse anti-vinculin (Santa Cruz Biotechnology) antibodies followed by HRP-conjugated anti-rabbit IgG or anti-mouse IgG (all from Sigma-Aldrich) and visualized with the ECL Western blotting substrate (Thermo Fisher Scientific), using a ChemiDoc Touch Imaging System (Biorad).