Ecl western blotting systems kit

Western blotting was performed as described previously [23] (link). The primary rabbit anti-mouse and rabbit anti-human α11 antibodies [29] (link) were used at a dilution of 1∶500, and the mouse anti-β-actin antibody at a dilution of 1∶5000. The secondary goat anti-rabbit and goat anti-mouse HRP-conjugated antibodies (Santa Cruz Biotechnology) were applied at a dilution of 1∶5000. Chemiluminescence signals were developed using the ECL Western-blotting systems kit (GE Healthcare) and photographed using the ChemiDoc XRS device and the Quantity One 1-D Analysis Software (Bio-Rad).

The cells cultured in monolayers were washed with phosphate-buffered solution (PBS, Sigma-Aldrich, St Louis, MO, USA) lysed in SDS-sample buffer (Bio-Rad, Oslo, Norway, Cat# 1610791) with 3% of 2-β-mercaptoethanol (Sigma-Aldrich, Cat# M7154) and sonicated using a Vibra-Cell™ ultrasonic processor (Sonics and Materials, Newtown, CT, USA). The cell lysates were subjected to (6% acrylamide) SDS-PAGE electrophoresis after boiling for 5 min., and the proteins were transferred to PVDF membranes using the iBlot^® system. The membranes were blocked with 5% non-fat dry milk (Marvel, UK) in Tris-buffered saline containing 0.1% Tween20 (TBS-T), incubated with primary mouse anti-human α11 antibody Mab 210F4 [70 ] or rabbit monoclonal anti-human α2 (EPR 5788, Abcam, Cambridge, MA, USA, Cat# ab133557) or mouse monoclonal anti-human α1 antibody (R&D Systems, Minneapolis, MN, USA, Cat# MAB 5676) and anti-β-actin (AC-74, Sigma-Aldrich, Cat# A5441) overnight at 4 °C. Following the incubations, the membranes were washed in TBS-T three times for 10 min and incubated with goat anti-mouse- or goat anti-rabbit-HRP-conjugated secondary antibodies for 1 h at room temperature. The membranes were developed while using the ECL™ western blotting systems kit (GE Healthcare) and photographed using the ChemiDoc XRS device and the Quantity One 1-D Analysis Software (Bio-Rad).