Odyssey protein molecular weight marker

Tumor lysates prepared for RPPA were also used for Western blot. Briefly, 20μg protein from each sample was used for 10% polyacrylamide gel electrophoresis, followed by transferring onto nitrocellulose membrane (Biorad, Cat. # 162–0115), blocking with Odyssey Blocking Buffer (PBS) (LI-COR, Lincoln, NE, Cat. # 927–40000) at room temperature for 1h, incubating with primary antibodies at room temperature for 1h and Infrared (IR)-labeled secondary antibodies (IRdye800CW goat anti-rabbit IgG, Cat. 92532211) at room temperature for 1h. Membranes were imaged and quantified using Odyssey Infrared Imaging System and its application software Version 3.0.30 (LI-COR). Newblot Nitro Striping Buffer (LI-COR, Cat. #928–40030) was used for antibody striping and subsequent probing with a different antibody. All first antibodies used are from Cell Signaling Technology (CST): anti-Phospho-MEK1/2 (Ser217/221) antibody (Cat. #9121, 1:1000); anti-MEK1/2 antibody (Cat. #9122, 1:1000); anti-Phospho-p44/42MAPK (Erk1/2) (Thr202/Try204) antibody (Cat. # 4377, 1:1000); and anti-p44/42 MAPK (Erk1/2) antibody (Cat. #9102, 1:1000). Odyssey Protein Molecular Weight Marker (LI-COR, Cat. 928–40000) was used as molecular weight reference.

All the fractions were diluted with 10x SDS-PAGE reducing sample buffer to a final concentration of 1x and denatured at 95°C for 5 min. Samples were run either along with eE2FL marker (std) and Odyssey Protein Molecular Weight Marker (Li-Cor) (L) on 4-20% Bis-Tris precast gels (Bio-Rad). Proteins were then transferred to PVDF membranes using a Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked by Intercept (PBS) Blocking Buffer (LI-COR) for 1 hour at 37°C followed by incubating the blot with a 1:1000 dilution of purified 2C1 mouse antibody (against HVR1 of HCV J6 E2 produced in Dr. Arash Grakoui’s laboratory) and incubated overnight at 4°C. Primary antibody dilution was prepared in Odyssey Blocking Buffer in PBS with 0.1% Tween 20 (Sigma-Aldrich). The secondary antibody, IRDye 800CW Goat anti-Mouse IgG (Li-Cor), was used in 1:10,000 dilution. The Western blot was scanned using Li-Cor Odyssey software (version 3.0). Fluorescence signals of top fractions were background corrected and measured by the Image Studio Lite software (version 5.2.5). The fluorescence intensities were exported in excel format and histograms were prepared.