Anti raf

After 24 h drug treatment, total protein was extracted using lysis buffer contained 4% SDS, protease inhibitor cocktail and phosphatase inhibitor (Roche, US). Equal amount of total proteins was resolved using denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blot. Antibodies used in WB analyses include anti-p-MYPT1 (Cell Signaling, Cat. No.4563), anti-p-MLC (Cell Signaling, Cat.No.3671), anti-MLC (Cell Signaling, Cat.No.3672), anti-MYPT1 (Cell Signaling, Cat. No.2634), anti-p-Raf (Abcam, Cat. No. ab135559), anti-Raf (Abcam, Cat. No. ab137435), anti-p-ERK (Santa Cruz, Cat. No. sc-16,982), anti-ERK (Santa Cruz, Cat. No. sc-292,838), anti-Ras(Q61L) antibody (NewEast Biosciences, Cat. No. NEBA10195) and anti-β-actin (Santa Cruz, Cat. No. sc-130,656). Immunoblots shown in the accompanying figures are representative of three independent experiments.

Total‐cell protein was extracted using RIPA buffer with protease inhibitors and phosphatase inhibitor (Life Technologies). The BCA Protein Assay Kit (Beyotime Bio‐ technology) was used to quantify the protein concentration. An equal amount of protein was loaded onto 8%–12% SDS‐PAGE and then transferred to PVDF membrance (Hercules bio rad). After incubation with 5% skimmed milk at room temperature for 1 h, the membrance was incubated with the following antibodies: anti‐BMAL1, anti‐E‐cadherin, anti‐N‐cadherin, anti‐vimentin, anti‐β‐catenin, anti‐phospho‐MEK, anti‐phospho‐ERK1/2 (Cell Signaling Technology), anti‐p38, anti‐JNK, anti‐c‐Myc, anti‐RAF, and anti‐Ki67 (Abcam). Anti‐GAPDH mouse monoclonal antibody (Cell Signaling Technology) was used as load control. The ECL system was used for blots (Amersham Biosciences).