2.8. PYK2 Activation and Western Blot

Cells were cultured on BSA, VCAM-1 or fibronectin for 15 and 30 min, harvested and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors. PYK2 activation was analyzed by immunoblot by utilizing the anti-phosphorylated-PYK2 (Tyr-402) antibody. The blots were then stripped and reprobed with anti-PYK2 antibody to ensure equal loading. The bands corresponding to phosphorylated PYK2 and total PYK2 were subjected to densitometry quantification with the Molecular Imager^® Gel Doc™ XR System from Bio-Rad Laboratories (Mississauga, ON, Canada). Uncropped western blots for Figure 7 can be found from Supplementary Figure S3.