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. 2008 May 30;283(22):15003–15014. doi: 10.1074/jbc.M801621200

FIGURE 1.

FIGURE 1.

TGFβ increases CRP2 expression in VSMCs. A, TGFβ induces CRP2 protein expression. VSMCs were exposed to TGFβ (10 ng/ml), and protein extracts were harvested at the indicated time points. Total protein was also extracted from unstimulated control cells. Western blot analyses were performed using 20 μg of total protein/lane. After electrophoresis, proteins were transferred to nitrocellulose membranes and incubated with a polyclonal primary antiserum specific for CRP2-(91–98) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The blot was visualized with enhanced chemiluminescence and exposed to film. Blots were subsequently probed for α-tubulin for normalization. CRP2 protein induction is expressed relative to control without TGFβ stimulation at 0 h. Values are mean ± S.E. of three experiments. B, up-regulation of CRP2 mRNA by TGFβ. VSMCs were treated with TGFβ (10 ng/ml) for the indicated times. Northern blot analysis was performed with 10 μg of total RNA/lane. After electrophoresis, RNA was transferred to nitrocellulose filters and hybridized with a random primed 32P-labeled mouse CRP2 cDNA probe that hybridized to a 1.0-kb CRP2 message. The blots were subsequently hybridized with a 32P-labeled 18 S oligonucleotide to verify loading. The signal intensity of each RNA sample hybridized to the CRP2 probe was divided by that hybridized to the 18 S probe. The normalized intensities were expressed relative to control without TGFβ treatment at 0 h. Values are mean ± S.E. of 3–5 experiments. C, TGFβ treatment reduces wild type but not Csrp2–/– VSMC migration in response to PDGF-BB. Wild type (+/+) and Csrp2–/– (–/–) VSMCs were serum-starved for 24 h, treated without or with TGFβ (10 ng/ml) for 12 h and then plated in triplicate in 6-well transwell plates for migration assays using PDGF-BB (10 ng/ml) as a chemoattractant. Cells migrating through the filters after 2 h were quantified and normalized to the cell number of wild type without TGFβ treatment. Values are mean ± S.D. of two experiments.