Fig. 5. TGFβ1 inhibits expression of Rad51 protein by affecting its degradation but not its mRNA expression. Expression of endogenous Rad51 protein was evaluated by western blotting (A) and by immunoprecipitation from ³⁵S-labelled cells (B). Mv1Lu cells were treated with TGFβ1 (5 ng/ml) for 2 and 24 h, and total cell lysate was subjected to SDS–PAGE and western blotting (A), or to immunoprecipitation (B) with Rad51-specific antibodies. Migration of Rad51 is shown by arrows. For control of equal loading, expression of α-tubulin was determined using the same cell extracts (for western blotting; A). (C) Rad51 mRNA expression was evaluated by northern blotting of total RNA prepared from cells treated as in (A) and (B), with a human Rad51 probe. Equal loading of RNA was evaluated by re-probing the same membrane with GAPDH probe, as shown. Migration positions for 18S and 28S rRNA are shown. (D) Inhibition of proteasomal degradation by treatment of cells with MG-132 (10 µM), lactacystin (10 µM) or ALLN (12.5 µg/ml) for 6 h (for MG-132) or for 8 h (for lactacystin and ALLN) before cell lysis, blocks TGFβ1-dependent decrease in Rad51 protein expression. Cells were treated with TGFβ1 (5 ng/ml) as indicated. Rad51 protein expression was evaluated by western blotting. Cytokeratin expression was used for normalization of equal loading, as α-tubulin expression in Mv1Lu cells was found to be sensitive to the MG-132 treatment. Migration positions of Rad51, α-tubulin and cytokeratin are shown. (E) TGFβ1 stimulates ubiquitylation of Rad51. Mv1Lu cells were treated with TGFβ1 (5 ng/ml) or not, as indicated. After cell lysis, immunoprecipitation (IP) with anti-ubiquitin or anti-Rad51 antibodies was performed, followed by western blotting (IB) with anti-Rad51 or anti-ubiquitin antibodies, as indicated. Migration positions of unubiquitylated (Rad51) or ubiquitylated Rad51 [Rad51-Ub(n)] are shown. Representative experiments of four (for A, C and D) and three (for B and E) performed, are shown.