Fig 3.

REF/Aly-binding correlates with ORF57 activity. (A) Immunoprecipitation of REF/Aly is abolished in the Fl-ΔReBD mutant. (Lanes 1 and 2) Western analysis demonstrating similar expression levels of the Fl-ORF57 and Fl-ΔReBD proteins. Endogenous β-actin served as a loading control. (Lanes 3 to 8) Western blot analysis of 10% input and 100% pellets from a coimmunoprecipitation experiment. Whole-cell extracts from HEK293 cells transiently expressing either Fl-ORF57, Fl-ΔReBD, or an untagged ORF57 were used for immunoprecipitation with anti-Flag–agarose in the presence of RNase. Western blotting (WB) was performed with REF/Aly or Flag antibodies as indicated. (B) (Top) Schematic diagram of the PAN-WT and PANΔENE expression constructs. Transcription is driven by the PAN RNA promoter, and constructs contain the PAN RNA polyadenylation signal (black box). (Bottom) Northern analysis comparing the effects of Fl-ORF57 or Fl-ΔReBD on the accumulation of full-length PAN RNA (WT) or PANΔENE (ΔENE). Membranes were probed with PAN RNA or control probes, as indicated. The loading control detects RNA from a cotransfected plasmid that expresses the murine mgU2-19/30 small Cajal body RNA (scaRNA) (11, 75) (C) Quantification of the Northern blot data. Each PAN RNA value was first normalized to the loading control, and all values are relative to the corresponding no-ORF57 sample. The error bars show standard deviations (n = 3); the P values are from a two-tailed, unpaired Student's t test of the indicated result with the matching no-ORF57 data set.