SM22 suppresses NF-κB signal pathways in VSMCs under inflammation condition.

PAC1 SMC cells were transfected with either SM22 expression plasmid or its empty vector control (Ctrl) plasmid followed by the treatment of LTβR-Fc (a LTβR agonistic antibody that activates LTβR-mediated NF-κB signal pathways) for 24 hours. (A) SM22 overexpression suppressed the transcription of proinflammatory markers by qPCR assays. n = 4. (B) SM22 overexpression suppressed the expression of proinflammatory proteins ICAM-1 and SDF-1 by WB assays. Quantification of protein levels after normalizing to GAPDH is shown on the right. n = 3. (C) SM22 overexpression suppressed luciferase activities of NF-κB site-luc reporter by luciferase assays. n = 3. (D) SM22 overexpression suppressed the expression of NF-κB protein p65, p50 and p52 in the nucleus by WB assays using the nuclear protein extract (NPE). Quantification of protein levels after normalizing to LAMIN B (a nuclear protein marker) is shown on the right. n = 3. (E) SM22 overexpression modulated the expression of key components of NF-κB canonical and non-canonical signal pathways in the cytoplasm by WB assays using the cytoplasmic protein extract (CPE). Quantification of protein levels after normalizing to GAPDH in WB is shown on the right of the representative WB images. n = 3. The molecular weight markers are indicated on the left side of the images. Note: *, **, and *** indicate p<0.05, p<0.01, and p<0.001 respectively vs. the control (Ctrl).