Fig. 5.

PRKD2 silencing interferes with the G1/S transition complex. U87MG (left panels) and GM133 (right panels) cells were transfected with nontargeting (S) and PRKD2 targeting (P5) siRNA. C = control, untreated cells. One representative immunoblot out of 3 independent experiments is shown. (A) Analysis of phosphorylated and total CDK2 and CDK4 proteins was performed by immunoblotting in cytosolic and nuclear fractions 3 days post transfection. Lamin A/C and β-actin were used as loading controls. (B) Phosphorylation of pRb on Ser⁷⁸⁰, Ser⁷⁹⁵, and Ser^807/811 and expression of total pRb was determined by immunoblotting in nuclear and cytoplasmic fractions. Lamin A/C and β-actin were used as loading controls. (C) CREB was analyzed for phosphorylation on Ser¹³³ by immunoblotting, and total CREB was used as loading control. The phospho-antibody used also detects the phosphorylated form of CREB-related protein ATF-1. The lower line shows c-Myc expression in control and silenced cells. (D) On day 4, (U87MG) and 6 (GM133) post transfection target gene expression was analyzed by qPCR using validated primer pairs. HPRT1 was used as housekeeping gene. Relative gene expression of target genes is presented in relation to scrambled RNAi. Results represent mean ± SD from 3 biological replicates (***P < .001). Gene expression ratios were calculated by REST as described in Materials and Methods.