Figure 7.

RNAi knockdown of dCDK12, hCDK12, or hCDK13 in cultured cells. (A) Mock and RNAi (dsRNA targeting exons E2, E4, or E7 of dCDK12 = CG7597) treatments were carried out for 48 h on cultured Drosophila S2 cells, extracts were prepared, and Western blot analysis was performed using rabbit affinity-purified anti-dCDK12 IgG. Goat affinity-purified anti-dRpb2 IgG was used to assess sample loading. Size standards are in the mw lane, and values in kilodaltons indicated at left. (B) Mock and RNAi treatments and analysis were as in A, except that the phospho-CTD (PCTD) of dRpb1 was detected using mouse anti-Ser2P mAb (H5). Again, dRpb2 was a loading control. (C) Mock and RNAi treatments and analysis were as in B, except that the PCTD of dRpb1 was detected using mouse anti-Ser5P mAb (H14). (D) Mock and siRNA treatments were performed on HeLa R-19 cells, extracts were prepared after 3 d, and Western blot analysis was carried out using rAP anti-hCDK13 IgG. The siRNAs used targeted hCDK13 (si-13), hCDK12 (si-12), or both (si-12 + 13); control siRNAs (si-con) have no homology with any known mammalian genes. The mw lane contains molecular weight markers, and the value is indicated at left. The blot was also reacted with anti-β-actin to show equal loading of extracts. (E) Mock and siRNA treatments were performed as in A, extracts were prepared after 3 d, and Western blot analysis was carried out using rAP anti-hCDK12 IgG and anti-β-actin. (F) Mock and siRNA treatments were performed on HeLa R-19 cells as in A, and Western blot analysis was carried out using rat anti-Ser2P (3E10) and mouse anti-non-phospho CTD (8WG16) mAbs. After analysis of Rpb1, the blot was reprobed with anti-β-actin antibodies. Note that the intensity of IIo in the si-control as detected by anti-Ser2P (lane 2) is usually the same as in the mock treatment (lane 1).