Las 4000 luminescent image analyzer with ccd camera

To examine the effect of α1(IV) or α2(IV) chains on FAK phosphorylation in TIG-1-20 cells, after 48 h of treatment with α1(IV) or α2(IV) chain-specific siRNA or control siRNA, FBS was added to the treatment medium at a concentration of 0.5%. At 30 min after adding FBS, cells were washed with PBS and lysed. Also, to investigate the replenishment effect of α1(IV) and α2(IV) chains on FAK phosphorylation, after 24 h of treatment with α1(IV) and α2(IV) chain-specific or control siRNA, siRNA treatment medium was replaced by serum-free culture medium obtained from TIG-1-20 cells treated with control siRNA or siRNA against both α1(IV) and α2(IV) chains (medium including or not including α1(IV) and α2(IV) chains, respectively), and then these siRNA-treated TIG-1-20 cells were cultured for 48 h, washed with PBS, and lysed. Phosphorylated FAK and total FAK in TIG-1-20 cells were assessed by western blotting with antibodies against phosphorylated FAK (Tyr397) and total FAK at a dilution of 1:1000 (Cell Signaling Technology, Danvers, MA, USA). Immunoreactivity on blots was detected with the LAS-4000 Luminescent Image Analyzer with CCD Camera (Fujifilm).