Anti p mek

Cells and tissues were lysed for 2 hours in RIPA lysis solution freshly prepared with a protease inhibitor (Roche). The concentration of extracted proteins was determined with a bicinchoninic acid protein determination kit (enhanced) (P0010, Beyotime, Shanghai, China). The extracted proteins were then separated by molecular size on SDS‐PAGE gels, followed by transfer to polyvinylidene fluoride membranes, which were incubated at 4 °C overnight with primary antibodies (1:200–1:1000) dissolved in Tris‐buffered saline containing Tween 20 containing 5% skimmed milk powder. The antibodies were as follows: anti‐TRPC1 (sc‐133076, Santa Cruz Biotechnology), anti‐HIF‐1α (abs130612, Absin), anti‐MEK (2352S, Cell Signaling Technology), anti‐p‐MEK (ab96379, Abcam), anti‐Erk (ab32537, Abcam), anti‐p‐Erk (4370S, Cell signaling technology), and anti‐GAPDH (21612, Signalway Antibody). Afterwards, the membrane was washed with Tris‐buffered saline containing Tween 20 and incubated with the corresponding secondary antibodies for 2 hours at room temperature. All the secondary antibodies were from Abcam. All blots were detected with an ECL substrate (180‐5001, Tanon, Shanghai, China), and images were captured with Image Lab Software (Bio‐Rad).

RPMI 1640 and FBS (Fetal Bovine Serum) were purchased from Thermo Fisher (Waltham, MA); anti-CCNY, anti-GAPDH, anti-ERK1/2, anti-MEK, anti-pERK1/2, anti-cyclin E and anti-pMEK antibodies were obtained from Abcam (Cambridge, England, UK); Muse^® Count & Viability Kit and Muse™ Cell Cycle Kit were from Millipore (Bedford, MA, USA); CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS) was bought from Promega (Madison, WI, USA); Bisbenzimide Hoechst 33342 were purchased from Sigma (St. Louis, MO).

RIPA Buffer (9800, Cell Signaling, Danvers, MA) was used to lyse and extract total proteins from A375 cells. After the centrifugion, protein samples were separated by SDS-PAGE to isolate the supernatant fractions. Subsequently, proteins were transferred onto the polyvinylidene fluoride (PVDF) membranes and blocked with 5% milk for 1 hr. Next, PVDF membrane was incubated with the following primary immunoblotting antibodies: anti-KIT(1:1000 dilution), anti-K-Ras (1:1000 dilution), anti-c-Raf (1:1000 dilution), anti-p-MEK (1:1000 dilution), anti-MEK (1:1000 dilution), anti-p-ERK (1:1000 dilution), anti-ERK (1:1000 dilution) and anti-GAPDH (1:1000 dilution) (Abcam, Cambridge, UK) at 37°C for 2 hr. Then, PVDF membranes were washed for at least 3-5 times and co-incubated with HRP-conjugated secondary antibody for 1 hr at room temperature. ECL detection reagents from Amersham Pharmacia Biotech, (Tokyo, Japan) were used to visualize immunocomplexes. Image Pro software was applied to calculate the intensity of protein bands.