Figure 2. Interdependence of SSRP1 and SPT16 levels: RNAi-mediated knock-down (KD) of either SSRP1 or SPT16 leads to reduced levels of both proteins. (A–C) Western blot analysis of SSRP1, SPT16, and β-actin protein levels in lysates of cells with KD of SSRP1 or SPT16: (A) HeLa cells transduced with lentiviral shRNA constructs targeting SSRP1 (three different shRNAs), SPT16 (two different shRNAs), or GFP (control) followed by puromycin selection; (B) HeLa and HT1080 cells transduced with a lentiviral tet-on regulated shRNA construct targeting SSRP1 and treated with doxycycline (Dox) for the indicated periods of time; (C) MCF10A, MCF7 and MBA-MB-231 cells transduced with lentiviral shRNA constructs targeting SSRP1 (#3), SPT16 (#2), or GFP (control) followed by puromycin selection as in (A). (D and E) SSRP1 and SPT16 protein levels are reduced earlier than their corresponding mRNAs upon siRNA-mediated KD of the opposite subunit.(D)RT-PCR (upper three panels) and western blot analysis (lower 3 panels) of SPT16 and SSRP1 mRNA and protein levels, respectively, in HT1080 cells at the indicated times after transfection of control siRNA, siSSRP1, or siSPT16.(E)Levels of SSRP1 and SPT16 mRNA were measured in HeLa cells at different time points after transfection of the indicated siRNAs using qPCR. Mean of 3 replicates +/− standard deviation.