Association of RHA with collagen mRNAs is LARP6-dependent. (A) Knockdown of LARP6 in HLFs. HLFs were transduced with adenovirus expressing control shRNA (SC, lane 1) or LARP6-specific shRNA (LARP6, lane 2). Expression of endogenous RHA, LARP6, and tubulin was analyzed by Western blot. (B) Knockdown of LARP6 decreases association of RHA with collagen mRNAs. (Top panel) Control shRNA (Sc, lanes 1,3) or LARP6-specific shRNA (LARP6, lane 2) was expressed in HLFs, and IP was done with anti-RHA antibody (lanes 1,2) or anti-fibronectin antibody (lane 3). The IP samples were analyzed for pull-down of collagen α1(I), collagen α2(I), and actin mRNA by radiolabeled RT-PCR. (Bottom panel) Analysis of mRNAs in the input material. (C) Analysis of collagen mRNA in the IP by real-time PCR. The data are presented as means and standard errors of the mean from three independent experiments. (D) LARP6 enhances association of RHA with collagen mRNAs. HEK293 cells were transfected with LARP6 (lane 1) or control protein RBMS3 (lanes 2,3). IP was performed with anti-RHA antibody, and the IP material was analyzed by RT-PCR for presence of collagen α1(I) and collagen α2(I) mRNAs by radiolabeled RT-PCR. (Bottom panel) Analysis of mRNAs in the input material. (E) Analysis of collagen mRNA in the IP by real-time PCR. Error bars, ±1 standard error of the mean (SEM). (F) Analysis of proteins in the input material. Endogenous RHA and loading control actin (ACT), HA blot with transfected LARP6 and RBMS3 (CON).
