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. 2010 Apr 15;21(8):1409–1422. doi: 10.1091/mbc.E09-06-0451

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© 2010 by The American Society for Cell Biology

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Figure 8. — Knockdown of h3/acidic CaP decreases phospho-ERK2 levels in NIH-3T3 cells. NIH-3T3 cells were either transfected with nontargeting siRNA-control (lane 1) or siRNA against h3/acidic CaP (siRNA-aCaP; lane 2). Seventy-two hours after transfection, whole cell extracts were generated and examined by Western blotting. The membrane was stained with specific antibodies against (A) phospho-l-caldesmon (pCaD); (B) ERK1, phospho-ERK1 (pERK1), ERK2, phospho-ERK2 (pERK2); and (C) h3/acidic calponin (aCaP), neutral calponin (h2CaP) and tubulin-α as indicated. (D) Densitometric measurement of Western blots, derived from three independent experiments, was performed. Total protein levels were normalized to tubulin-α and the ratio of phosphorylated to unphosphorylated ERK2 protein was calculated and graphed. Note that the decrease of phosphorylated ERK2 (p = 0.00002) in h3/acidic CaP knockdown cells is highly statistically significant.