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. 2009 Dec 28;120(1):303–314. doi: 10.1172/JCI40364

Figure 5. Stat3 and p65 coassociate and regulate Rantes expression in VSMCs.

Figure 5

(A) VSMCs were transfected with reporter plasmids containing the native Rantes promoter (Co) or mutated plasmids (M1 or M2) designed to abolish putative NF-κB–binding site activity. Location and sequence of putative NF-κB–binding sites and mutations are indicated. Comparisons are Co p21–/– versus Co WT, or M1 or M2 versus Co for WT and p21–/–. pGL2 indicates WT VSMCs transfected with pGL2 plasmid containing no promoter. n = 3 for all groups; **P < 0.005, ***P < 0.001, ****P < 0.0001. (B) shRNA p65Kd in p21 VSMCs reduced mRNA expression of p65 (left panel, n = 3 both groups; **P < 0.01) and Rantes (right panel, n = 4 both groups; *P < 0.05) compared with Co shRNA. (C) Tnf-α treatment for 4 hours increased Rantes mRNA expression in WT Co VSMCs (n = 6 for unstimulated, n = 7 for stimulated; P < 0.0005), while p65Kd and Stat3Kd VSMCs exhibited attenuated Tnf-α–induced Rantes mRNA expression compared with Tnf-α–treated Co VSMCs (p65Kd: n = 3, *P < 0.05; Stat3Kd: n = 4, **P < 0.005). Data are shown as mean + SEM. (D) IP followed by WB in WT Co and Stat3Kd VSMCs (input protein levels as indicated). IP with anti-Stat3 then WB with anti-p65 (upper left) and IP with anti-p65 then WB with anti-Stat3 (lower left) revealed coassociation of p65 and Stat3. IP with anti-p65 then WB with anti-p21Cip1 (upper right) revealed coassociation of p65 with p21Cip1, which was virtually abolished in Stat3Kd VSMCs.