eIF3f facilitates hMSH4 stabilization. (A) The effect of RNAi-mediated down-regulation of eIF3f on the levels of endogenous hMSH4 was analyzed in A549 cells. A mixture of eIF3f sh-1 and sh-2 RNAi constructs was used for transient transfection, and cells were collected and analyzed by immunoblotting at 48 hrs post-transfection. Immunoblotting with α-tubulin was used as a loading control. (B) Stable expression of eIF3f and hMSH4 in 293T/eIF3f and 293T/eIF3f-hMSH4 cell lines (from selected single clones). (C) Western blotting analysis of the levels of HDAC3, hRad51, and VBP1 expression in 293T, 293T/eIF3f, and 293T/eIF3f-hMSH4 cells. (D) Effects of reduced eIF3f expression on the levels of hMSH4 in the stable cell line 293T/eIF3f-hMSH4. Reduction of eIF3f expression was achieved by transient transfection of eIF3f RNAi constructs. (E) Nuclear and cytoplasmic distribution of hMSH4 and eIF3f proteins in response to IR. 293T/eIF3f-hMSH4 cells treated with 1 or 10 Gy IR were fractionated at 6 hrs post-treatment and the levels of hMSH4 and eIF3f in the nuclear and cytoplasmic fractions were determined by immunoblotting. α-tubulin was used as a marker for the cytoplasmic fraction.