Anti human galnt6 polyclonal antibody

Western blot was performed as described previously [14] (link). Protein bands were visualized by ECL detection reagents (GE Healthcare). The primary antibodies used in this study were as follows: anti-human ER-α monoclonal antibody (1:400, Santa Cruz), anti-human GALNT6 polyclonal antibody (1:500, Sigma-Aldrich), anti-Flag M2 monoclonal antibody (1:1000, Sigma-Aldrich), anti-HA monoclonal antibody (1:1000, Roche), and anti–β-actin monoclonal antibody (1:10,000, Sigma-Aldrich). The secondary antibodies were goat anti-rabbit or anti-mouse IgG-HRP antibodies (1:10,000-1:30,000, Santa Cruz). For the detection of O-glycosylated proteins, we performed lectin blotting using biotinylated Vicia villosa agglutinin (VVA) lectin (1:1000, Vector Laboratories) and Streptavidin-HRP (1:10,000, Thermo Scientific).

Western blot was performed as described previously [17] (link). Finally, protein bands were visualized by ECL or ECL prime detection reagents (GE Healthcare). The primary antibodies used in this study were: anti-human GRP78 polyclonal antibody (1:1000, Santa Cruz), anti-human GALNT6 polyclonal antibody (1:1000, Sigma-Aldrich), anti-Flag M2 monoclonal antibody (1:1000, Sigma-Aldrich), anti-HA monoclonal antibody (1:1000, Roche), anti-PARP-1 antibody (1:1000, Santa Cruz), anti-caspase 7 (1:1000, Cell signaling), anti-PERK (1:1000, Cell signaling), anti-IRE1α (1:1000, Cell signaling), anti-ATF6 (1:1000, Cell signaling), and anti-β-actin monoclonal antibody (1:10,000, Sigma-Aldrich). The secondary antibodies were goat anti-rabbit, anti-rat, and anti-mouse IgG-HRP secondary antibodies (1:10,000~ 1:30,000, Santa Cruz). Intensity of protein band was quantified by ImageJ software as previously described [8] (link).

Immunocytochemistry was performed as previously described [14] (link). For siRNA knockdown, at 72 hours after adding siRNA, the cells were fixed. For cell-permeable peptide treatment, at 24 hours after seeding of cells, peptides were added and then incubated for 1 or 2 hours (1 hour for T47D cells and 2 hours for MCF7 cells), and then the cells were fixed. The primary antibodies used in immunocytochemical analysis included anti-human GALNT6 polyclonal antibody (1:500, Sigma-Aldrich), anti–ER-α antibody (1:400, Santa Cruz), and Alexa Fluor Phalloidin 594 (1:40, Thermofisher Scientific).The secondary antibodies were Alexa Fluor 488 anti-Mouse IgG antibodies (1:1000, Life Technologies) and Alexa Fluor 594 anti-Rabbit IgG antibodies (1:1000, Life Technologies). Finally, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and examined by TCS SP5 Confocal Laser Scanning Microscope (Leica Microsystems).