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. 2007 Sep 20;26(20):4368–4379. doi: 10.1038/sj.emboj.7601845

Figure 4.

Figure 4

NFATc1 and NFκB are O-GlcNAc-modified. (A) Western blot analysis of O-GlcNAc modifications of NFκB-p65 after immunoprecipitation (5 × 107 Jurkat cells each) using anti-NFκB antibodies against p65 subunits of NFκB and irrelevant antibodies (anti-c-myc) as a negative control. To visualize the background bands of the antibodies used for IP, we did IPs omitting the lysate. Positions of NFκB subunits as that of IgGH are indicated. (B) Western blot analysis of O-GlcNAc modifications after immunoprecipitation (5 × 107 Jurkat cells each) using an anti-NFATc1 antibody or irrelevant antibodies (anti-c-myc) as a negative control. To visualize the background bands of the antibodies used for IP, we did IPs omitting the lysate. Positions of NFATc1 proteins are indicated. (C) Western blot analysis of NFκB-p65, NFATc1 and O-GlcNAc modifications after immunoprecipitation (5 × 107 Jurkat cells each) using anti-O-GlcNAc antibodies (RL-2 and HGAC85). Positions of NFκB-p65, NFATc1 and IgGH are indicated at the right side of the panels. To visualize the background bands of the antibodies used for IP, we did IPs omitting the lysate. Antibodies used in the immunoprecipitation reactions are given at the top, whereas the antibodies used in the Western blots are at the left side. (D) Western blot analysis of O-GlcNAc modifications after immunoprecipitation (5 × 107 BJAB cells each) using an anti-NFATc1 antibody. The Western blot was developed with or without preincubation of the anti-O-GlcNAc antibody in 500 mM GlcNAc. (E) Western blot analysis of O-GlcNAc modifications after immunoprecipitation (5 × 107 BJAB cells each) using an anti-NFκB-p65 antibody. The Western blot was developed with or without preincubation of the anti-O-GlcNAc antibody in 500 mM GlcNAc.