Rabbit anti myc antibody

Huh7 cells were cultured on a 20-mm glass bottom dish (NEST) and transfected with the indicated plasmids. Then the cells were washed three times with PBS, fixed with 4% paraformaldehyde–containing PBS for 20 min, and permeabilized with 1% Triton X-100–containing PBS for 20 min. After being blocked with 2% bovine serum albumin–PBS for 1 h, the cells were incubated with primary antibody diluted in antibody dilution buffer overnight at 4°C.After being washed three times with PBS, the cells were incubated with second antibody for 1 h and washed with PBS for three times. Then the cells were treated with DAPI (Beyotime) for 10 min, washed three times with PBS, and observed with a confocal microscope (Olympus). The following primary and second antibodies were used: mouse anti-NS5A antibody 9E10 was kindly provided by Chaoyang Li (Wuhan Institute of Virology, Chinese Academy of Sciences, China); mouse anti-Flag antibody was purchased from Beyotime; rabbit anti-Myc antibody was purchased from Proteintech; TRITC-conjugated or FITC-conjugated second antibodies were purchased from Pierce.

Protein samples were separated on SDS-PAGE gels and then electroblotted onto nitrocellulose membranes. The nitrocellulose membranes were blocked for 1 h in 5% non-fat milk in TBST (10 mM Tris, pH 7.5, 200 mM NaCl, and 0.2% Tween 20) followed by incubation with primary antibodies: mouse anti-GFP antibody (Cat. 66002-1-Ig, Proteintech, Rosemont, IL, United States), rabbit anti-GFP antibody (Cat. ab290, abcam, Cambridge, United Kingdom), mouse anti-FLAG antibody (Cat. F3165, Sigma), rabbit anti-FLAG antibody (Cat. 20543-1-AP, Proteintech), rabbit anti-Myc antibody (Cat. 16286-1-AP, Proteintech), mouse anti-β-Actin antibody (Cat. 66009-1-Ig, Proteintech), rabbit anti-CDK2 antibody (Cat. 10122-1-AP, Proteintech), or rabbit anti-pCDK2 antibody (Cat. ab194868, abcam). Two secondary antibodies were used: goat anti-mouse antibody (Cat. ab216776, IRDye 680RD, abcam) and goat anti-rabbit antibody (Cat. ab216773, IRDye 800RD, abcam). Signals were captured by an Amersham Typhoon (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed by ImageQuant TL (GE Healthcare).