Anti myc antibody 4a6

HEK293T cells were transfected with constructs pTW351 (B4GAT1dTM) using FuGENE 6 (Roche Applied Science, Indianapolis, IN). The construct contains an IRES-puromycin resistance cassette and stable cell lines were selected in medium containing puromycin (1 µg/ml, InvivoGen, San Diego, CA). Expression and secretion of B4GAT1dTM protein into the culture medium was confirmed by immunoblotting with anti-Myc antibody 4A6 (Millipore). The stable cell lines obtained in this way were adapted to serum-free medium 293SFMII (Invitrogen) and cultivated in CELLine bioreactors (CL1000, Argos Technologies, Elgin, IL).

The anti-myc antibody (4A6) was obtained from Millipore (Billerica, MA). The anti-CDK5 antibody (C-8) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-actin antibody was purchased from Sigma (St Louis, MO). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated swine anti-rabbit IgG were purchased from Dako (Glostrup, Denmark). Alexa Fluor 488-conjugated anti-mouse IgG was purchased from Invitrogen (Carlsbad, CA). 4′,6-Diamidino-2-phenylindole (DAPI) was from Dojindo (Kumamoto, Japan). Phos-tag acrylamide was obtained from Wako (Osaka, Japan). Protein A/G Sepharose was purchased from GE Healthcare (Buckinghamshire, UK).