Whole cell lysate extracts were prepared M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, 78503) supplemented with 1X Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, 78447). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, 23225). SDS-PAGE was performed using the Bolt system (Invitrogen) including 1X LDS sample buffer and reducing agent, 4-12% Bis-Tris Plus gels, and MES running buffer. Proteins were transferred to PVDF membranes using the iBlot2 transfer system (Invitrogen). Membranes were blocked in Superblock T20 (PBS) Blocking Buffer (Thermo Fisher, 3716) and incubated in primary antibody (listed below) in 5% BSA in TBST overnight at 4°C. Secondary antibodies (listed below) were diluted in 0.1% TBST. Membranes were imaged using the Bio-Rad ChemiDoc Imager and bands were quantified using Image Lab software (Bio-Rad).
Primary antibodies were diluted as follows: GALNT1 (NBP1-81852, lot A115764, Novus) 1:1000; GALNT7 (NBP2-39021, lots R89578 and 000010086, Novus) 1:1000, GAPDH (D16H11; 51745, lot 4, Cell Signaling Technologies) 1:1000, Actin (Ab-5, 612652, lot 6176513, BD Transduction Laboratories) 1:10,000. Secondary antibodies: Goat anti-Mouse Ig (554002, lot 5247553, BD Pharmingen) 1:10,000, Goat anti-Rabbit IgG (15015, lot 04318009, Active Motif) 1:10,000.
Primary antibodies were diluted as follows: GALNT1 (NBP1-81852, lot A115764, Novus) 1:1000; GALNT7 (NBP2-39021, lots R89578 and 000010086, Novus) 1:1000, GAPDH (D16H11; 51745, lot 4, Cell Signaling Technologies) 1:1000, Actin (Ab-5, 612652, lot 6176513, BD Transduction Laboratories) 1:10,000. Secondary antibodies: Goat anti-Mouse Ig (554002, lot 5247553, BD Pharmingen) 1:10,000, Goat anti-Rabbit IgG (15015, lot 04318009, Active Motif) 1:10,000.