P mek ser217 221

Western blotting was performed as previously described39 (link). The conditions and duration of treatments of the cells before protein isolation are described in the text and the figure legends. Primary antibodies included p-ERK Thr204/205, ERK, p-MEK Ser217/221, MEK, anti-HA (clone C29F4) and GAPDH (all from Cell Signaling Technology, Danvers, MA), BRAF (H-145) and CRAF (E-10) (both from Santa Cruz biotechnology, Santa Cruz, CA), anti-MYC and anti-actin (Sigma Aldrich, St. Louis, MO). Secondary antibodies were HRP conjugates (Santa Cruz biotechnology, Santa Cruz, CA). Densitometry of bands was performed by using ImageJ program.

All melanoma cells were washed with ice-cold phosphate buffered saline (PBS) twice and lysed with RIPA buffer containing phosphatase and protease inhibitors (all from Sigma Aldrich, St. Louis, MO). Protein extracts were separated with SDS-PAGE in 4-12% tris-glycine gels and transferred to immun-blot PVDF membrane. After blocking for 1 hour in PBS containing 0.1% Tween 20 and 5% nonfat milk or 5% bovine serum albumin (BSA) in PBS, the membrane was exposed to various primary antibodies overnight, followed by secondary antibodies conjugated to horseradish peroxidase. ECL-Plus kit (Amersham Biosciences Co, Piscataway, NJ) was used to check immunoreactivity and blots were scanned using a Typhoon scanner (Amersham Biosciences Co.). Primary antibodies included pERK Thr202/Tyr204, total ERK, pMEK Ser217/221, total MEK, pAKT Ser473, total AKT, pRSK, total RSK, beta-actin (all from Cell Signaling Technology, Danvers, MA).

Cells were lysed with 50 mM Tris-HCl (pH 7.5) containing 1 mM EDTA, 50 mM NaF, 150 mM NaCl, 1% Triton X-100, 10% glycerol plus 1% phosphatase and protease inhibitor cocktails (Bimake, Houston, TX, USA). Proteins were separated using SDS-PAGE and were transferred onto nitrocellulose membrane (Bio-Rad Laboratories, CA, USA). The following primary antibodies were used: anti-PCSK9 antibody (85813, Cell Signaling Technology, Danvers, MA, USA), pERK1/2 (T202/Y204) (Cell Signaling Technology, 9101), ERK1/2 (Cell Signaling Technology, 4696), pAKT (Ser473) (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 2920), pMEK Ser217/221 (Cell Signaling Technology, 9121), MEK (Cell Signaling Technology, 4694) and anti β-tubulin (Santa Cruz Biotech sc-166729, Dallas, TX, USA). Specific LI-COR secondary antibodies IRDye^® 680RD Donkey anti-Goat IgG, IRDye^® 800CW Donkey anti-Mouse IgG, or IRDye^® 680RD Donkey anti-Rabbit IgG (LI-COR, Lincoln, NE, USA) were used before detection with Odyssey^® CLx Imaging System (LI-COR). Image analysis was performed using Image Studio ™ Lite software (LI-COR).