shRNA against Cyclin T2 expressed from a lentiviral vector specifically and efficiently depletes CyclinT2 protein. (A) HeLa cells infected at an m.o.i. of five with lentiviral vectors expressing a shRNA against Cyclin T2 (shRNA T2), Cyclin T1 (shRNA T1) or a control shRNA against a mismatch sequence in Cyclin T1 (shRNA MM). Cultures were analyzed five days post infection by flow cytometry. HeLa cells not treated with any shRNA were used as a control for flow cytometry analysis. The lentiviral vectors express a GFP marker protein. The unfilled region represents GFP background level in non-treated HeLa cells. The percentages of GFP positive cells are indicated. (B) Total RNA was extracted from HeLa cells transduced with shRNA T2 or shRNA MM after five days post infection and analyzed Cyclin T2, Cyclin T1 mRNA by quantitative real time RT-PCR. The fold-change is indicative of the transcript levels in the shRNA T2 treated cells relative to the shRNA MM treated cells after normalization to housekeeping gene, β-actin levels. (C) Immunoblot analysis of cell extracts prepared from HeLa cells transduced for five days with shRNA T2, shRNA T1 or shRNA MM lentiviral vectors. Untransduced HeLa cells (Mock) were used as control. The immunoblots were performed to analyze the levels of Cyclin T2, Cyclin T1, Cdk9, HEXIM1 and β-actin proteins. The band intensity was quantified using ImageJ and presented below each panel.