Anti gapdh antibody

Lysates from cells were prepared in radioactive immunoprecipitation assay buffer and treated with protease inhibitor cocktail. Equal amounts of proteins were added to Laemmli’s buffer (Bio-Rad, Hercules, CA), heated for 5 min at 95℃, and loaded into a 15-wells 4% to 12% Bis-Tris gel (Invitrogen, Carlsbad, CA). Proteins were then transferred to PVDF membranes (Bio-Rad, Hercules, CA), blocked with 5% nonfat dry milk in Phosphate Buffered Saline with Tween-20 (PBST), and incubated with primary antibody anti-SORT1 (1 µg/ml; (Abcam, Cambridge, MA; ab16640) and secondary antibody anti-Rabbit conjugated to horseradish peroxidase (1:5,000; Abcam; ab6721). Anti-GAPDH antibody (1:5000, Trevigen, Gaithersburg, MD) was used as loading control. Protein expression was detected by using Lumminata Forte (Millipore, Billerica, MA), gel images were obtained using a G:BOX Chemi image analyzer (Syngene, Frederick, MD), and densitometric analyses were performed through the ImageJ software (imagej.nih.gov).

Protein samples (50 μg) with or without glycosidase treatment were separated by 5–20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene fluoride (PVDF) membranes. After incubating with 2% enhanced chemiluminescence (ECL) blocking reagent (GE Healthcare, Buckinghamshire, UK), the membranes were incubated with goat anti-DINE antibody (1:500; Santa Cruz Biotechnology) at 4 °C overnight. The membranes were repeatedly washed then incubated with horseradish peroxidase-conjugated anti-goat IgG secondary antibody (1:5000; Vector, Burlingame, CA, USA). Anti-GAPDH antibody (1:5000; Trevigen, Gaithersburg, MD, USA) was used for the control experiments. Each set of experiments was repeated at least three times to confirm results.

HEK293T cells were purchased from American Type Culture Collection (Manassas,VA), cultured in Dulbecco’s modified Eagle’s media (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Korea, Seoul, Korea), and transfected with plasmid using Lipofectamine 2000 (Thermo Fisher Scientific Korea) according to the manufacturer’s instructions. Subsequently, the cells were lysed with M-PER mammalian protein extraction reagent (Thermo Fisher Scientific Korea) at 48 h post-transfection, and processed for Western blotting as described previously [27 (link)]. The antibodies used were anti-FLAG antibody (1:2000, Sigma-Aldrich, catalog number F1804), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (1:2000, Trevigen, Gaithersburg, MD, 2275-PC-100), HRP-conjugated goat anti-mouse antibody (1:4000, Santa Cruz Biotechnology, Dallas, TX, sc-2005), and HRP-conjugated goat anti-rabbit antibody (1:4000, Santa Cruz Biotechnology, sc-2004). Band intensity on the Western blots was analyzed using ImageJ.