Sc 9090

Proteins were extracted from cells (80% confluence) with lysis buffer (150 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 0.1% SDS, 1% sodium deoxycholate, 1% Nonidet P-40, 1X protease inhibitor cocktail (Roche), 50 mM NaF, 2 mM Na₃VO₄, 40 mM β-glycerophosphate and 1 mM dithiothreitol), separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, and incubated with primary antibodies (viz. goat anti-ITGB1, rabbit anti-ITGB4 and rabbit anti-LAMR antibodies (sc-9936, sc-9090 and sc-20979, respectively; Santa Cruz Biotechnology)) (1:1000 dilution) followed by rabbit anti-goat or goat anti-rabbit horseradish peroxidase (HRP)-conjugated IgG secondary antibodies (sc-2768 and sc-2004 respectively; Santa Cruz Biotechnology). Clarity Western ECL reagent (BioRad, Hercules, CA, USA) was employed to obtain chemiluminescent signals recorded by a G-Box Chemi XL system (Syngene, Cambridge, UK). Actin or GAPDH used to normalize gel loading was detected with rabbit anti-actin (SAB5600071; Sigma-Aldrich, St. Louis, MO, USA) (1:2000) or goat anti-GAPDH (sc-48166; Santa Cruz Biotechnology) (1:2000) primary antibodies followed by the appropriate secondary antibodies as described above. The band intensity was quantified using Fiji software [45 (link)].