Fig. 8

HOXA1 transcriptionally activates RelA. A Real-time qPCR assay for NF-κB RelA expression in POV-PC treated VSMCs. B Western blot assay for phospho-p65^ser536 and nuclear p65 expression. C EMSA for HOXA1 binding to NF-κB RelA promoter in POV-PC treated VSMCs. D 293 T cells were co-transfected with a GFP-HOXA1 expression vector and a RelA-promoter reporter plasmid for 72 h. The cell lysates were prepared for luciferase assay. E The cell lysates of VSMCs were subjected to oligonucleotide pull-down assay with biotinylated double-stranded oligonucleotides containing RelA-promoter as probe. Nonspecific oligonucleotide sequences were applied as a negative control. The retrieved protein was collected and analysed by Western blot with anti-HOXA1 antibody. F ChIP-qPCR assay was performed in VSMCs with anti-HOXA1 antibody and non-immune IgG was used as an internal control. Immunoprecipitated DNA was amplified by PCR using RelA-promoter primers. Negative regions (regions downstream of the RelA-promoter) was adopted as an amplification control. Data are expressed as mean ± SD (n = 3). Ordinary one-way ANOVA followed by Tukey's multiple comparison test was used to calculate the P value in panel A, B, D. Two tailed unpaired t-test was used to calculate the P value in panel F. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001