Figure 4
From: MafK positively regulates NF-κB activity by enhancing CBP-mediated p65 acetylation
MafK deficiency inhibits NF-κB DNA binding.
(a) HepG2 cells were transfected with MafK or control siRNA and after 48 h, cells were treated with LPS (1 μg/mL) followed by measurement of IκBα degradation and phosphorylation in lysates after 15, 30, 60, 180 and 300 min. (b) HepG2 cells were transfected with MafK or control siRNA and after 48 h, cell were stimulated with LPS for 30 min. Nuclear and cytoplasmic fractions were analysed by immunoblotting. (c) HepG2 cells were stimulated with LPS for 6 h, followed by chromatin immunoprecipitation using anti-p65, anti-Acetyl-p65 and normal rabbit IgG (control) antibodies. Chromatin immunoprecipitation was analysed using primers specific to the IL-8 and TNFα promoter regions. (d) MafK deficiency suppressed p65 acetylation but not affected phosphorylation. HepG2 cells were transfected with MafK or control siRNA, then stimulated with LPS as indicated time. (e) HepG2 cells were transfected with MafK or control siRNA for 48 h and then treated with LPS and incubated for 6 h, collected and lysed in immunoprecipitation lysis buffer. Whole-cell lysates were immunoprecipitated with human anti-p65 antibody and probed by western blotting with rabbit anti-CBP antibodies. (f) HepG2 cells were transfected with MafK siRNA, or MafK siRNA and MafK plasmid. After 48 h, cells were treated with LPS and incubated for 6 h; the cells were collected and lysed in immunoprecipitation lysis buffer. Whole-cell lysates were immunoprecipitated with human anti-p65 antibody and probed by western blotting with rabbit anti-CBP antibody.
