Skip to main content
. 2016 Aug 5;7(35):57186–57196. doi: 10.18632/oncotarget.11083

Figure 4. Low glucose condition promotes O-GlcNAcylation of ATG4B.

Figure 4

A. SH-SY5Y/HA-ATG4B cells were cultured in normal (4.5 mg/ml glucose) or low glucose condition (Low glu, 1 mg/ml glucose). After 24 h, total O-GlcNAc proteins were pull-down using sWGA-agarose antibody and the immune-complex was analyzed with Western blotting (WB) with anti-HA antibody. And the input lysates were further detected with anti-HA and anti-LC3 antibodies. B. For endo-IP assay, SH-SY5Y cells were incubated in normal or low glucose condition. And whole O-GlcNAc proteins were pull-down using sWGA-agarose antibody or IgG antibody. The immune-precipitates were further assessed with Western blotting with anti ATG4B antibody. C. SY5Y/HA-ATG4B cells transiently transfected with either scrambled siRNA (Sc) or siRNA against OGT were cultured in low glucose condition for 24 h. Then the cell lysates were analyzed by Western blotting with anti-HA antibody after immunoprecipitation with anti-sWGA-agarose antibody. Knock down of OGT protein by siRNA was confirmed by immunoblotting with anti-OGT antibody. D. SH-SY5Y/ATG4B cells were incubated in low glucose (1 mg/ml glucose) for 24 h. And the cells were recovered with normal high glucose media (4.5 mg/ml glucose) or not for additional 3 days. O-GlcNAc proteins were pull-down using sWGA-agarose antibody and immuneblotted with anti-HA antibody.