Anti o glcnac rl2

The method for determination of OGG1-specific O-GlcNAcylation was described previously (28 (link)) with modifications. Briefly, 500 µg of total protein samples from FLAG-OGG1 expressing hSAECs were subjected to immunoprecipitation (IP) using FLAG antibody or β-actin (1:500 dilution; sc-1615, Santa Cruz Biotechnology) was used as internal control at 4°C overnight. 30 µL of Protein A/G Magnetic beads (Millipore, MAGNA0017) blocked with normal rabbit IgG (Santa Cruz Biotechnology, SC-2027) were added on next day and incubated for 3 h. Immunoprecipitants were washed extensively and analyzed by Western blotting using anti-O-GlcNAc (RL2) (Thermo Fisher Scientific, MAI-072) and anti-FLAG antibodies. Images were acquired with the Amersham Blot and Gel Imager 680 (Global Life Sci. Sol. Marlborough, MA).

For nutrient treatment experiments, cells were seeded at 4.5 × 10⁵ cells/well in 6-well plates overnight then treated with normal DMEM (10% FBS, 4.5 g/L glucose) supplemented with doxycycline for 48 hours, to induce endogenous OGT knockdown and expression of our exogenous construct. Samples were then treated with the nutrient condition detailed in the results. Following low nutrient treatment, cells were washed with PBS and harvested by centrifugation. Cell pellets were lysed with RIPA total lysis buffer (150 mM NaCl, 1% nonidet-P40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris-HCl, pH 7.4) supplemented with protease inhibitor cocktail (Millipore Sigma #P8340) and 10 μM Thiamet-G. Cell debris was removed by centrifugation at 16,000 g for 15 min at 4°C. Samples were separated on an 8% SDS-PAGE gel and detected by western blot using antibodies: anti-O-GlcNAc (RL2, ThermoFisher scientific #MA1–072), anti-OGT (Santa Cruz #sc-74546), anti-OGA (Santa Cruz #sc-376429), anti-Flag (Millipore Sigma #F1804), anti-cMyc (Millipore Sigma #C3956), and anti-β-Actin (ThermoFisher scientific #MA1–140).

Cells were washed with ice-cold PBS 3 times and lysed with NET lysis buffer. The buffer was composed of 150 mM NaCl, 50 mM Tris, 1 mM EDTA, and 1% Nonidet P-40, had a pH of 7.4, and was supplemented with a protease inhibitor cocktail (Roche). Lysates were boiled in the SDS sample buffer for 5 min and then subjected to Western blot analysis. The following antibodies were used:
Anti-O-GlcNAc (RL2: Thermo Fisher Scientific, #MA1-072), Anti-SMAD4 (Santa Cruz, #sc-7966), Anti-SMAD2/3 (Cell Signaling Technology, #3102S), Anti-OGT (DM-17: Sigma, #O6264), Anti-OGA (Abcam, #ab124807), Anti-GAPDH (Millipore, #M171-3 or Santa Cruz, #sc-32233), Anti-FLAG (MBL, #M185-3L), Anti-Ubiquitin (Santa Cruz, #sc-8017), Anti-HA (Santa Cruz, #sc-805 or Cell Signaling Technology, #C29F4), Anti-GSK-3β (Cell Signaling Technology, #27C10), Anti-Phospho-GSK-3β (Ser9) (Cell Signaling Technology, #D3A4), Anti-Phospho-GSK-3β (Tyr216) (Abcam, #ab75745), Anti-Phospho-threonine (Santa Cruz, #sc-5267), Anti-c-myc (Santa Cruz, #sc-47694).
For immunoprecipitation and sWGA purification, total cell lysates were incubated with antibody-bound beads or agarose-conjugated succinylated wheat germ agglutinin (sWGA, Vector Laboratories) overnight at 4 °C. Beads were then washed 5 times with lysis buffer and bead-bound proteins were eluted by boiling the beads for 5 min in the SDS sample buffer.