Anti rabbit igg horseradish peroxidase hrp linked secondary antibody

The protein concentrations within the fractionated sample loaded onto the gel were normalized by total protein content using the bicinchoninic acid (BCA) assay (Thermo Scientific Pierce, Waltham, MA). Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Gels were rinsed in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol), and the proteins transferred onto a nitrocellulose membrane in transfer buffer for 1.5 h at 100 V at 4°C. After transfer, membranes were blocked with 5% milk in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.1% Tween) for 1 h and washed with PBST four times for 5 min each time. Primary rabbit antiacetyllysine antibody (Cell Signaling, Danvers, MA) was diluted 1,000-fold in 5% bovine serum albumin (BSA), added to the membranes, and incubated in the cold room with shaking. The membrane was washed 4 times with PBST for 5 min each time and incubated for 1 h in the dark at room temperature with anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling, Danvers, MA) diluted 2,000-fold in 5% milk. The membrane was washed 4 times with PBST for 5 min each time, incubated in ECL blotting substrate (Abcam), and imaged in the Protein Simple machine (Bio-Techne) (13 (link), 19 (link)).

Samples for Western blots were collected, prepared, and analyzed as described previously [31 (link),32 (link)]. Briefly, 661W cells were harvested and lysed in a Tris lysis buffer (in mM): 50 Tris, 1 EGTA, 150 NaCl, 1% Triton X-100, 1% β-mercaptoethanol, 50 NaF, and 1 Na₃VO₄, pH 7.5. Samples were separated on 10% sodium dodecyl sulfatepolyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked in 3% BSA in tris buffered saline Tween 20 (TBST) at room temperature for 1 h and incubated in primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated in anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary antibody (1:1000, #7074S, Cell Signaling, Beverly, MA, USA) at room temperature for 1 h. The blots were visualized using Super Signal West Pico/Femto chemiluminescent substrate (#34078/#34096, ThemoFisher). Band intensities were quantified using Image J (National Institutes of Health; NIH, Bethesda, MA, USA). The primary antibodies used in this study were anti-ELK1 (1:500, #9182S, Cell Signaling) and anti-β-actin (1:2000, #8457L, Cell Signaling). The band intensities of ELK1 were normalized to those of β-actin.

BGB‐283, compound C, and pimasertib were synthesized in‐house and exceeded a purity of 99% as measured by proton nuclear magnetic resonance (HNMR), liquid chromatography–mass spectrometry (LC‐MS), and high‐performance liquid chromatography (HPLC). Compounds were purchased from following source: vemurafenib, WuXi AppTec (Shanghai, China); selumetinib, PD‐0325901, and trametinib, BioChemPartner (Shanghai, China); and RO5126766, Active Biochem (Kowloon, Hong Kong). Stock solutions of compounds were prepared in dimethyl sulfoxide. Antibodies used were obtained commercially from the following sources: anti‐B‐RAF (SC‐5248), Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐C‐RAF (610152), BD Biosciences (San Jose, CA, USA); antibodies to MEK (9122), MEK1 (2352), phospho‐MEK1/2 (Ser217/221) (9154), ERK (4695), phospho‐ERK1/2 (Thr202/Tyr204) (4370), GAPDH (2118s), and anti‐rabbit IgG horseradish peroxidase (HRP)‐linked secondary antibody, Cell Signaling Technology (Danvers, MA, USA); and anti‐mouse IgG HRP‐linked secondary antibody (A0168), Sigma‐Aldrich (St. Louis, MO, USA). BALB/c nude mice (female) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All procedures involving animals were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of BeiGene.