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. 2010 Aug 16;31(10):1293–1302. doi: 10.1038/aps.2010.96

Figure 4.

Figure 4

ATRA promotes KLF4 phosphorylation and its interaction with p300 by activating p38 MAPK and JNK signaling. (A) VSMCs were treated with 10 μmol/L ATRA for the indicated times, cell lysates were immunoprecipitated with antibody to phosphoserine and immunoblotted with antibody to KLF4. (B) VSMCs were treated with 10 μmol/L ATRA for the indicated times, cell lysates were immunoblotted using phospho-specific antibodies to detect phosphorylated JNK and phosphorylated p38, respectively. Blots for total protein (JNK or p38) are also shown. (C) VSMCs were pretreated with JNK inhibitor SP600125 (20 μmol/L) or p38 inhibitor SB203580 (20 μmol/L) for 2 h before exposure to ATRA (10 μmol/L) for 1 h. The phosphorylated KLF4 was assessed as described above. (D) VSMCs were pretreated with SP600125 (20 μmol/L) or SB203580 (20 μmol/L) for 2 h before exposure to ATRA (10 μmol/L) for 1 h. Interaction of KLF4 with p300 was examined by CoIP and Western blot analysis with the indicated antibodies as described above. (E) VSMCs were pretreated with SP600125 (20 μmol/L) or SB203580 (20 μmol/L) for 2 h before exposure to ATRA (10 μmol/L) for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (F) VSMCs were transfected with expression vectors for GFP-N1, GFP-KLF4, or its phosphorylation-deficient mutants, GFP-KLF4 (S415A), GFP-KLF4 (S444A), and GFP-KLF4 (S470A), after 24 h of transfection, cells were treated with or without ATRA (10 μmol/L) for 1 h. Cell lysates were immunoprecipitated with antibody to phosphoserine and immunoblotted with antibody to KLF4. (G) VSMCs were transfected with expression vectors for GFP-N1, GFP-KLF4, or GFP-KLF4 (S470A), after 24 h of transfection, cells were treated with or without ATRA (10 μmol/L) for 1 h. Interaction between KLF4 and p300 was examined by CoIP and Western blot analysis with the indicated antibodies as described above. (H) VSMCs were transfected with expression vectors for GFP-N1, GFP-KLF4, or GFP-KLF4 (S470A), after 24 h of transfection, cells were treated with or without ATRA (10 μmol/L) for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (I) Luciferase activity assays. A293 cells were transfected with the mfn-2 promoter-reporter plasmid (containing nucleotides −441 to +15 of the mfn-2 promoter), along with different combinations of expression plasmids for GFP-N1, GFP-KLF4 or its phosphorylation-deficient mutant GFP-KLF4 (S470A), and p300 for 24 h, and then treated with ATRA (10 μmol/L) for 24 h in the presence or absence of SP600125 (20 μmol/L) or SB203580 (20 μmol/L). The bars represent the means±SEM from three independent experiments. bP<0.05 vs GFP-N1 group.