Fig. 1. Inhibition of HBP expression by RNA interference. (A) Top: Schematic drawing showing the location of the shRNA target sequences in the 810 nucleotide HBP open reading frame. Boundaries of sequences coding for domains involved in histone RNA binding (RBD) and RNA processing (RP) and for the putative translation stimulation domain (T) are indicated (Wang et al., 1996; Dominski et al., 1999; Sanchez and Marzluff, 2002). The position of the sequences coding for residues responsible for the control of HBP stability (St) are also shown (Zheng et al., 2003). Note that the drawing is not to scale. Middle: Schematic diagram showing the structure of the RNA interference cassette inserted into the pGEM-T Easy cloning site. Shown are pGEM-T Easy flanking regions, Sp6 RNA polymerase promoter (SP6), U6 RNA promoter (U6P) and termination signal (TTTTT). U6 promoter and terminator flank the inverted repeat coding for the shRNAs. MluI: restriction sites used for the excision of the cassettes and the subcloning into pEGFP-C3. Bottom: Sequences of the shRNAs used to inhibit HBP expression by RNAi. The control hairpin cHP deviates at two positions (indicated in lower case letters) from HP1. (B) HeLa cell extract was analysed by western blotting using anti-HBP serum. Strips were probed with both primary and secondary antibody, secondary antibody only, or with primary antibody preincubated with the antigenic peptide, and the secondary antibody. The HBP position is marked by the arrow; ^* marks a major non-specific band with the mobility corresponding to a 38 kDa protein. The positions of molecular mass standards are indicated. (C) HeLa cells were transfected with either pGEM-U6pHP1 or pGEM-U6p or mock-transfected (no DNA). Protein samples prepared 24 hours after transfection were analysed by western blotting using anti-HBP and subsequently anti-tubulin antibodies as loading control. Blots were stripped between subsequent probings. ^*as in B. rHBP is recombinant human HBP. (D) HeLa cells were transfected with pGEM-U6pT, pGEMU6pHP1 or pGEM-U6HP3 and treated as described for B.