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. Author manuscript; available in PMC: 2010 Jan 1.
Published in final edited form as: Cancer Res. 2009 Jan 1;69(1):65–74. doi: 10.1158/0008-5472.CAN-08-0377

Figure 2. PKC alpha inhibition induces changes in MAPK activity and regulates cell cycle genes.

Figure 2

A) Decreased ERK activity in SQ20B cells with PKCα inhibition. SQ20B cells were transfected with control (siControl) or PKCα (siPKCalpha) siRNA. After 48 hours in serum-free media, cells were incubated with increasing concentrations of PDBu (left panels) or serum (right panels) for 30 minutes. Whole cell lysates were probed for phosphorylated and total ERK, and blots were quantified as described in Experimental Procedures. Graphs represent quantification of the ratio of phospho-ERK to total ERK protein. Representative experiments are shown for at least two independent experiments. B) Gene array heat map of cell cycle genes in the SQ20B cell line with decreased expression at 24 hours upon exposure to 100nM Gö6976 (left panel). Right panel shows RT-PCR expression of selected cell cycle genes at 0 (black bars), 12 (gray bars), and 24 (white bars) hours shown as fold change normalized to untreated control. C) Protein immunoblotting and immunoprecipitation of selected cell cycle genes upon exposure to 100nM Gö6976 at 0, 12, and 24 hours. SQ20B cells were serum-starved for 48 hours, followed by treatment in serum with 100 nM Gö6976 for 0, 12, and 24 hrs. Total protein was extracted from cells and immunoblotted for cyclin E, Cdk2, E2F2, cyclin A, MCM6 and γ or α-tubulin as described in Experimental Procedures (left panel). Cell lysates were immunoprecipitated with an anti-Cdk2 antibody and the immunoprecipitates were examined by immunoblotting to detect cdk2-complexed cyclin E (cdk2 IP/cyclin E IB, right panel). Total protein lysates were also probed for cyclin E and cdk2 antibodies. Representative data from three separate experiments are shown. D) E2F 1-3 protein levels in SQ20B cells. SQ20B cells were serum-starved for 48 hours, followed by treatment in serum with 100 nM Gö6976 for 0, 12, and 24 hrs. Total protein lysates were probed with the respective antibodies. Lysate from untreated U2OS cells was used as a positive control for E2F1.