Anti dykddddk flag tag antibody

Co-immunoprecipitation assay and western blotting (WB) were performed as previously described45 (link). Lysates were prepared from HuH-7 cells transfected with plasmids of interest 48 hr after transfection. The antibodies used in co-immunoprecipitation were anti-Cx43 antibody (Sigma), anti-T7-tag antibody (Novagen), anti-HA-tag antibody, anti-DYKDDDDK (FLAG)-tag antibody (Wako Pure Chemical), and normal mouse and normal rabbit IgG (Santa Cruz Biotechnology). The antibodies used in WB were anti-Cx43 antibody (Sigma), anti-Hsc70 antibody (StressMarq), anti-T7-tag antibody (Novagen), anti-HA-tag antibody, anti-DYKDDDDK (FLAG)-tag antibody, anti-cMyc-tag antibody (Wako Pure Chemical), and anti-GAPDH antibody (Santa Cruz Biotechnology). The signals were measured by Image Quant LAS500 (GE Healthcare UK Ltd., Buckinghamshire, England).

DTT, dipyridyl, NH₄Cl, an anti-DYKDDDDK (FLAG) tag antibody, an anti-Myc tag antibody, leupeptin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Diamide, N-ethylmaleimide (NEM), methoxypolyethylene glycol (PEG) maleimide, fetal bovine serum (FBS), penicillin-streptomycin solution, and S-methyl methanethiosulfonate (MMTS) were obtained from Sigma-Aldrich (St. Louis, MO). (N-[6-(biotinamido)hexyl]-3’-(2’-pyridyldithio) propionamide (Biotin-HPDP) and streptavidin-agarose were purchased from Pierce Biotechnology (Rockford IL). An anti-HSC70 antibody was purchased from Novus Biologicals (Littleton, CO). An anti-BiP antibody was purchased from GeneTex, Inc. (Irvine, CA). Dimethyloxaloylglycine (DMOG), Diamide, and a horseradish peroxidase (HRP) Maleimide Conjugate were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). KOD plus neo DNA polymerase was purchased from TOYOBO (Tokyo, Japan). Revert AidTM M-MuLV Reverse transcriptase was purchased from MBI Fermentas (Vilnius, Lithuania).

HuH-7 cells and PANC-1 cells were plated on 35-mm glass covered dishes. After 20 hr of cell seeding, the cells were transfected with plasmids of interest. After 48 hr of transfection, the cells were fixed in 2% paraformaldehyde (PFA) in PBS for 1 hr. After fixation, the cells were permeabilized by incubation with 0.1% Triton X-100 in PBS for 10 min, blocked with 3% skim milk in PBS for 1 hr, and incubated with primary antibody in 3% skim milk solution overnight. Primary antibodies used were: anti-Cx43 antibody (Sigma, St. Louis, MO), anti-T7-tag antibody (Novagen, Madison, WI, USA), anti-cMyc-tag antibody, anti-DYKDDDDK (FLAG)-tag antibody (Wako Pure Chemical, Osaka, Japan), anti-HA-tag antibody (Y11, Santa Cruz Biotechnology), and anti-p27 antibodies (C19, Santa Cruz Biotechnology). Secondary antibodies used were: Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 594 goat anti-mouse IgG (Invitrogen-Molecular Probes) in 3% skim milk solution for 1.5 hr. DNA was stained with TO-PRO3 iodide (Invitrogen-Molecular Probes) for 30 min. Cells were washed carefully with PBS (3 times, 10 min each time) after incubation with primary and secondary antibodies. After immunostaining, samples were mounted and analyzed by confocal microscope (FV1000, Olympus).