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. Author manuscript; available in PMC: 2016 Jul 12.
Published in final edited form as: Biochem J. 2015 Jul 14;470(2):255–262. doi: 10.1042/BJ20150610

Figure 2. CpOGAD298N can be utilized as a Far Western tool.

Figure 2

a) Immunoblots with anti-O-GlcNAc antibodies (RL2 and CTD110.6) or Far Western with GST-CpOGAD298N was performed on either naked or in vitro O-GlcNAcylated TAB1 (labels adjacent to the blots). The specificity of the signal detected was confirmed by deglycosylating O-GlcNAcylated TAB1 by pre-treatment with CpOGAWT. Pre-treatment as indicated above the blots were performed to probe the activity of all the CpOGA constructs.

b) Embryonically overexpressed DmOGT-HA was immunoprecipitated with HA antibody and immunoblotted with anti-O-GlcNAc antibodies (RL2 and CTD110.6) or subjected to Far Western with GST-CpOGAD298N. Treating the immunoprecipitate with CpOGAWT was used as a control.

c) HEK293 lysates without or with CpOGAWT pre-treatment were subjected to GST-CpOGAD298N Far Western. In addition, the GST-CpOGAD298N Far Western was performed in the presence of 0.5 M GlcNAc competition. The extreme right panel is an immunoblot of the same samples with anti-O-GlcNAc antibody, RL2.

d) HEK293 lysates without or with PNGase F/CpOGAWT pre-treatment were subjected to GST-CpOGAD298N Far Western. The right panel is an immunoblot of the same samples with anti-O-GlcNAc antibody, RL2.