(A) Immunoprecipitations were performed using anti-Gemin5 or control antibodies to analyze snRNAs associated with Gemin5 in cells treated with cyclohexmide (CHX) or anisomycin (ANS) and crosslinked with formaldehyde. Immunoprecipitated RNA levels were quantitated by RT-qPCR using specific primers. Experiments were done at least three times; a typical experiment is shown. Error bars indicate standard deviation of three replicates.
(B) Northern blots were performed on RNAs analyzed in panel (A) using snRNA specific probes. RNA species larger than mature snRNAs are indicated as pre-snRNAs. RNAs from the immunoprecipitation using anti-Sm antibody served as a reference for mature snRNAs. The blot was also probed for 5S and 5.8S RNAs as controls.
(C) Experiments were performed as in (A) except cells were treated with anisomycin (ANS), actinomycin D (ACT D) or a combination of the two for 4 hrs and RNA immunoprecipitations were performed using anti-SMN and anti-Gemin5 antibodies.