Click it o glcnac enzymatic labeling system

Chemoenzymatic labeling and biotinylation of proteins in total cell lysates were carried out as described previously (14 (link), 47 (link)). In brief, proteins (200 μg) were labeled utilizing the Click-iT O-GlcNAc Enzymatic Labeling System (Invitrogen). The permissive mutant β-1,4-galactosyltransferase (GalT) is responsible for the transfer of azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues on target proteins. Modified proteins were detected utilizing the Click-iT Biotin Protein Analysis Detection Kit protocol (Invitrogen). Biotinylated proteins were resolubilized in binding buffer (0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2). Appropriate amount of streptavidin resin (Thermo) was added to incubate with the mixture overnight at 4 °C. The streptavidin-bound complex was washed with binding buffer. Following the removal of supernatants, pellets were eluted by boiling with loading buffer (2% SDS, 10% glycerol, 2.5% 2-mercaptoethanol, 62.5 mM Tris–HCl, pH 6.8) and analyzed by WB.

Click-it O-GlcNAc Enzymatic Labeling System (Invitrogen C33368) is a method for modification in vitro of O-GlcNAcylated proteins. Proteins were enzymatically labeled by the permissive mutant B-1,4 galactosyltransferase (Gal T1 Y289L), which transfers azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues in the target proteins. A protein extract with no enzymatic treatment was used as a negative control, and α-crystallin, a protein with a low O-GlcNAcylation level (2–10%) was used as a positive control. Click-it O-GlcNAc enzymatic labeling was performed following the manufacturer's protocols. Labeled proteins were detected by Western blot with the Click-it Biotin Protein Analysis Detection Kit (Invitrogen C33372).

In vitro O-GlcNAcylation of TAB3 was detected as previously described [8 (link)]. The TAB3 (1 mM) was added into 20 μl reaction buffer (50 mMTris-HCl [pH = 7.5], 1 mM DTT, 12.5 mM MgCl₂) which contain 50 mM OGT and 1 mM UDP-GlcNAc. The reaction mixtures were incubated for 90 min at 37°C, stopped by adding loading buffer, resolved on Western blot with appropriate antibodies. To enzymatic label TAB3 at O-GlcNAc site, O-GlcNAcylated GST-TAB3 bound beads and was labeled using Click-iT^™ O-GlcNAc enzymatic labeling system (Invitrogen, Carlsbad, USA) and detected by Click-iT^™ biotin protein analysis detection kit (Invitrogen, Carlsbad, USA) following manufacturer's recommendation. β-elimination was performed overnight at 4°C using the GlycoProfile β-elimination kit (Sigma, St. Louis, USA) according to the manufacturer's instructions.