Anti myc antibody

Cells were fixed with cold acetone for 10 min. After washing, cells were incubated in PBS containing 0.3% Triton X-100 for 10 min and blocked with 1% BSA for 30 min. Then, cells were incubated with anti-Myc antibody (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., mouse, 1:500 dilution), and anti-HA antibody (Proteintech, rabbit, 1:100 dilution) at 4°C overnight. Following additional washes, cells were incubated with the appropriate secondary antibody (1:200; Beyotime Biotechnology) at room temperature for 1 h, and then stained with DAPI. Images were acquired using Zeiss Laser scanning confocal microscope (Lsm880 NLO).

Whole cell lysates were prepared for immunoblotting as described previously [23 (link)–25 (link)]. Equal amounts of total proteins (30 μg) were subjected to SDS-PAGE, transferred onto nitrocellulose, and immunoblotted with specific antibodies. The primary antibodies against phospho-STAT3 (Tyr705), STAT3, phospho-JAK2 (Tyr1007/1008), JAK2, PARP, MCL1, XIAP, Cox2 and iNOS were purchased from Cell Signaling Technology (Danvers, MA). Anti-β-Actin, anti-α-Tublin, anti–mouse IgG and anti–rabbit IgG horseradish peroxidase conjugated antibodies were purchased from Santa Cruz (Santa Cruz, CA). Anti-Myc antibody was purchased from Medical & Biological Laboratories (Tokyo, Japan). Anti-GAPDH antibody was purchased from Abgent (Suzhou, China).

Cell extracts were lysed in SDS buffer and a protease inhibitor cocktail (Roche Applied Science). Proteins were resolved by 10% SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to PVDF (polyvinylidene difluoride) membrane (Millipore) by electroblotting. Membranes were blocked in TBS containing 5% skim milk, and incubated with the following primary antibodies: anti-Galectin-1 (Abcam, Cambridge, MA) antibody, anti-V5 antibody (Life Technologies) and anti-Myc antibody (Medical & Biological Laboratories, Nagoya, Japan). The secondary antibodies for chemoluminescence detection were peroxidase-conjugated anti-mouse or anti-rabbit IgGs (Jackson ImmunoResearch Laboratories, West Grove, PA). Signal was obtained by enhanced chemoluminescence (Western Lightning Ultra, Perkin Elmer, Waltham, MA).