Anti o glcnac ctd110.6

IP and immunobloting experiments were performed as previously described (42 (link)). O-GlcNAc was enriched by treating the cells with Glu plus TMG as previously described (treated with 5 μM TMG for 24 h and 30 mM Glu for 3 h) (41 (link)). Antibodies used were as follows: anti-ULK1 (CST; catalog no.: 8054), anti-OGT (Santa Cruz; catalog no.: sc-32921), anti-O-GlcNAc (RL2) (Abcam; catalog no.: ab2739), anti-O-GlcNAc (CTD110.6) (Sigma; catalog no.: O7764), LC3B (CST; catalog no.: 2775S), and anti-STX17 (Merck; catalog no.: HPA001204).
Peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch. Blotted proteins were visualized using the ECL detection system (Amersham). Signals were detected by a LAS-4000 (Fujifilm) and quantitatively analyzed by densitometry using the Multi Gauge software (Fujifilm). All Western blots were repeated at least three times. Silver staining analysis was performed as described (41 (link)).

Proteins were run on 12% SDS-PAGE under reducing conditions. Gels were either stained with Coomassie blue or electroblotted onto a PVDF sheet. Blots were saturated with 5% (w/v) blotting-grade blocker (Bio-Rad) in TBS (Tris-buffered saline)-Tween [15 mM Tris, 140 mM NaCl, 0.5% (v/v) Tween] for 30 min. Primary antibodies were incubated overnight at 4°C. Mouse monoclonal anti-O-GlcNAc RL2 (ab2739) was used at a 1:1,000 dilution; mouse polyclonal anti-O-GlcNAc CTD 110.6 and anti-alpha tubulin DM1A (Sigma, St Louis Missouri, USA) antibodies were also used. The specificity of the RL2 antibodies was checked by co-incubation with 1 M free O-GlcNAc. Then, the membranes were washed three times for 10 min with TBS-Tween and incubated with anti-mouse IgG or IgM, HRP labeled secondary antibodies (Abcam, Cambridge, UK) at a 1:5,000 dilution. The membranes were washed three times for 10 min with TBS-Tween, and spots were detected by enhanced chemiluminescence with Hyperfilms (GE Healthcare, Chicago, USA). Three independent experiments were performed and images were captured using a Bio Rad Gel Doc imaging system and processed with Quantity One software. One representative blot is shown.