Validation of LARP4B target mRNAs. (A) Flag/HA-LARP4B was immunoprecipitated from cell extract of LARP4B overexpressing HEK293 cells using anti-Flag antibodies (lane 2). To control for specificity a cell line not overexpressing LARP4B was used (lane 4). Copurified RNA was isolated and analyzed by RT-PCR. mRNAs ranked high in the PAR-CLIP list could be confirmed as LARP4B targets while low ranked targets and nontargets could not be copurified. Western blot analysis demonstrates the efficiency of the LARP4B immunoprecipitation. (B) Endogenous LARP4B specifically interacts with target mRNAs. For immunoprecipitation of endogenous LARP4B an anti-LARP4B antibody was used (lane 2). A control immunoprecipitation was performed with pre-immune serum (PIS, lane 3). Endogenous LARP4B interacted with target mRNAs while the control RNA, RCOR1 mRNA, was not co-immunoprecipitated.
