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. 2012 Sep;32(18):3663–3680. doi: 10.1128/MCB.00406-12

Fig 2.

Fig 2

Expression of HsRAD51 K133 mutants reduces colony number and size. (A) Ratios of HAT-resistant colonies were compared for HsRAD51WT (WT) and the negative control (KS) to HsRAD51K133A (KA) and HsRAD51K133R (KR) with and without eGFP. Shown is the total number of HAT-resistant colonies observed from four experiments for the untagged Cre-mediated knock-in plasmids. Colony numbers are listed in sequential order from the first to the fourth experiment, and some of plasmids were not examined (shown by “ND” [not done]) for some experiments: HsRAD51WT, 337, 262, 106, and 208; KS, 337, ND, ND, and 123; HsRAD51K133A, 20, 26, ND, and ND; HsRAD51K133R, 84, 100, 18, and ND. The total numbers of HAT-resistant colonies observed from three experiments for the eGFP-tagged Cre-mediated knock-in plasmids are as follows: eGFP-HsRAD51WT, 52, 227, and 261; eGFP-HsRAD51K133A, 7, 19, and 20; and eGFP-HsRAD51K133R, 15, 75, and 76. AB2.2 cells are shown. (B) Surface area of HAT-resistant colonies after knock-in. The total numbers of colonies observed are as follows: HsRAD51WT, 8; KS, 8; HsRAD51K133A, 7; and HsRAD51K133R, 8. AB2.2 cells are shown. (C) RT-PCR on cells expressing untagged HsRAD51. AB2.2 cells are the control (Con). (D) Western analysis of nuclear extracts using anti-RAD51 antibody on cells expressing eGFP-HsRAD51. AB2.2 cells are the control. (E) Nonspecific eGFP-HsRAD51 and MmRad51 shRNA knockdown. The total colony numbers are shown. (F) MmRad51-specific shRNA knockdown. Three experiments were performed. The number of colonies that grow after stable transfection of shRNA plasmid is divided by the number of colonies that grow after stable transfection of empty vector for cells that express the following: eGFP-HsRAD51WT, 98/188, 103/180, and 99/176; eGFP-HsRAD51K133A, 14/118, 17/144, and 15/106; and eGFP-HsRAD51K133R, 15/110, 15/118, and 16/114. AB2.2 cells are shown. (G) Western analysis after no transfection (NT) or transfection with empty vector (EV) or shRNA for MmRad51. Two clones were observed for cells that express eGFP-HsRAD51WT in AB2.2 cells. α-HsRAD51, anti-HsRAD51; α-β-actin, anti-β-actin.