FIG 4.

Mutation of the LC8 binding site prevents recruitment of CITFA2 to promoters and its assembly into the CITFA complex. (A) Comparison of constitutive wild-type (WT), NDel, or 3Amut CITFA-HA expression in individual BF cell lines by immunoblotting, with TFIIB serving as a loading control. (B) Anti-HA ChIP assays in cell lines constitutively expressing wild-type or NDel CITFA2-HA. Precipitated DNA was analyzed using primer pairs which amplified the consensus BES promoter (BES prom), the consensus RRNA promoter (RRNA prom), and the β/α-tubulin intergenic region. Error bars represent one standard deviation, with asterisks indicating a Student's t test P value of <0.05. (C) Anti-HA coimmunoprecipitation with the same cell lines. Blots monitoring wild-type (WT) or NDel CITFA2-HA immunoprecipitation were probed to detect the coimmunoprecipitation of LC8, CITFA6, CITFA7, and, as a loading control, TFIIB. Note that IgG light chain (IgG l.c.) was detected at the top of the CITFA6 immunoblot. (D) Sucrose gradient sedimentation of whole-cell extracts, prepared from cell lines that constitutively express wild-type (WT), NDel, or 3Amut CITFA2-HA cell lines, was analyzed by immunoblotting fractions 4 to 20 using either anti-HA or anti-CITFA6 immune serum.