Fig. 3.

Co-localization of endogenous p32 with FBL and Nop52 in the nucleolus and Cajal bodies, and exclusion of p32 from the nucleolus by actinomycin D treatment. 293EBNA cells were fixed before (whole-cell view) or after (nuclear view) permeabilization and immunostained using an antibody against p32, FBL, FLAG, Nop52, p80-coilin, or SMN. p32, corresponding immunolocalization of endogenous p32. FBL, corresponding images stained for FBL indicating dense fibrillar component (DFC) of the nucleoli. FLAG-Nop52 and Nop52, corresponding images stained for FLAG-Nop52 and endogenous Nop52, respectively, indicating granular component (GC) of the nucleoli. p80-coilin, corresponding images stained for p80-coilin indicating Cajal bodies. SMN, corresponding images stained for SMN indicating Cajal bodies. Merge/DAPI for (A)∼(D), merge of p32 and DAPI, and FBL (A), or FLAG-Nop52 (B), or p80-coilin (C), or SMN (D). Merge/DAPI for (E), merge of Nop52 and DAPI, and p80-coilin. F, 293EBNA cells were treated with 2 μg/ml actinomycin D (ActD) for 2 h or 4 h, and stained for analysis by indirect immunofluorescence (p32 in green; B23 in red). Ethanol, treated with ethanol for 4 h. Merge/DAPI, merge of p32, B23, and DAPI. Bars indicate 10 μm. G, Total proteins from cells treated for longer periods with ActD analyzed by immunoblotting with the antibodies indicated at the left. Pol II (N-20), RNA polymerase II (antibody against hypo- and hyper-phosphorylated forms of Pol II).