Rabbit anti raf

The activation states of Raf, Erk and p53 in differentially treated HCECs were determined using Western blotting assays as previously described [29 (link)]. Briefly, four groups of differentially treated HCECs were harvested for Western blotting after coculturing with amoeba for 24 h. The cells were lysed in 100 μL lysis buffer (1 mL lysis buffer containing 20 μL phosphatase inhibitors, 20 μL protease inhibitor cocktail, 100 μL PBS, 5 μL NP-40 and 855 μL ddH₂O). The cell lysate (20 μg) was resolved by SDS-PAGE and transferred to PVDF membranes (Roche, Switzerland). We used the following primary antibodies purchased from Cell Signaling Technology (USA): rabbit anti-p-Raf (Ser338), rabbit anti-Raf, rabbit anti-p-Erk (Thr202/Tyr204), rabbit anti-Erk, mouse anti-p-p53 (Ser15) and mouse anti-p53. HRP-labelled goat anti-mouse and goat anti-rabbit secondary antibodies (Abcam, UK) and rabbit polyclonal to β-actin (Abcam, UK) were detected with Tanon™ High-sig ECL Western Blotting Substrate (Tanon, China), observed with an ECL detection system (Tanon, China), and the scanned images were quantified using Image-Pro Plus 4.5.1 software (Media Cybernetics, USA).