Click it glycoprotein detection kit

Notch-GFP-expressing trachea from n = 150 ND/HSD-fed white pupae were lysed in 220 μl homogenization buffer (125 mM NaCl, 50 mM HEPES, 5% NP-40, 1% TritonX-100, 100 μM TMG, pH 7.9). 210 μl supernatant per sample was collected, of which 10 μl was reserved as loading control. Then, supernatants were incubated with UDP-GalNAz and Gal-T1 (Y289L) enzyme from the Click-it GlcNAc enzymatic labeling system (Thermo Fisher, #C33368). In the enzymatic reaction, Gal-T1 transfers the azide-modified galactose (GalNAz) from UDP-GalNAz to GlcNAc-modified proteins in the supernatant. Subsequently, the azide-modified products were labeled by biotin using a Click-it glycoprotein detection kit (Thermo Fisher, #C33372). Streptavidin magnetic beads (Thermo Fisher, #88816) were used to capture the biotin-labeled GlcNAcylated proteins. The loading control and biotin-bound GlcNAcylated proteins were separated by SDS/PAGE on 4–15% Bis-Tris gels and followed by immunoblot analysis. Primary antibody: α-GFP (rabbit, 1:2000, Invitrogen, #A-11122). Secondary antibody: HRP-conjugated α-rabbit (goat, 1:5000, Abcam, #ab6721).

Labelling of O-GlcNAc-bearing proteins by GalNAz and biotin alkyne was done using the Click-it O-GlcNAc enzymatic labeling system and the Click-it Glycoprotein detection kit (Biotin alkyne) according to the manufacturer's instructions (Fischer Scientific) (18). Bovine α-crystallin was used as a positive control. After labeling, proteins were precipitated using the methanol/chloroform protocol and resuspended in 50 μL of Tris/HCl pH 8.0 containing 0.1% (w/v) SDS. 700 μL of enrichment buffer (1% (v/v) Triton X-100 and 0.1% (w/v) SDS in PBS) was added to the sample before incubating with 50 μL of avidin-coupled beads (1 h, 4°C). Avidin-bound proteins were collected, washed three times with enrichment buffer, resuspended in Laemmli buffer and boiled. Controls of labeling and enrichment followed the same procedure except that the chemical labeling with UDP-GalNAz was omitted.