2080 Nucleic Acids Research, 2005, Vol. 33, No. 7 Figure 1. (A) Schematic diagrams of Fxh and A2BP1 isoforms and summary of their subcellular distribution and splicing activities. The broken lines and broken squares indicate the excluded and untranslated sequences, respectively. The light blue region has a reading frame shifted from the equivalent white region. The relative activity of each isoform to the others are represented as numbers of +s; more ‘+’s are higher and is no activity. ND, not determined; nls, exogenous NLS-containing proteins; and wt, wild-type proteins. (B) Schematic diagrams of the exon–intron organizations of mouse Fxh and A2BP1 genes. Rectangles and solid lines in the diagrams indicate exons and introns. Zigzag lines of rectangles indicate undefined ends of exons. Exon and intron sizes are not drawn to scale. transcription/translation system (Promega) from pCS3 + MT constructs, which include the SP6 promoter. Binding reactions were carried out in a 10 ml mixture that contains 10 mM HEPES (pH 7.9), 2 mM MgCl2, 50 mM KCl, 5% glycerol, 0.5 mM DTT, 5 mg tRNA, 1 ml reticulocyte lysate reaction mixture and 15 fmol of probe, on ice for 20–30 min. An aliquot of 5 mg of heparin was added to the reaction 10 min before gel electrophoresis. The reaction mixtures were analyzed by elec- trophoresis in a 6% polyacrylamide gel using a 0.5· TBE buffer (Invitrogen). Cell culture and transfection The human retinoblastoma cell line Y79 was cultured and transfected with DNAs as described previously (14,26). Total amounts of transfected DNAs were adjusted to be con- stant by addition of the empty vector. Either Lipofectin (Invit- rogen) or Effectene transfection reagent (Qiagen) was used for the transfection. For stable transfection, the pCS3+MT expres- sion constructs were co-transfected with a plasmid carrying a neomycin resistant gene and selected by 0.2 mM geneticin (Invitrogen). For differentiation of Y79 cells, cells were plated on the poly-D-lysine-coated plates and then treated with 2.0–2.5 mM sodium butyrate for 4–5 days. HeLa cells were cultured as described and transfected with DNA using Effectene reagent (14,26). Immunoblot analysis Samples that required both protein and mRNA analysis were split upon harvesting. Total cell proteins were subjected to SDS–PAGE and blotted as described previously (26). The primary antibodies used are monoclonal antibodies to a myc-epitope (Invitrogen) and green ﬂuorescent protein (GFP) (Clontech). Binding of antibodies was detected with the SuperSignal System (Pierce) or ECL (Amersham). Immunofluorescent microscopy HeLa or Y79 cells grown in a four-chamber glass slide were transfected as described above. Cells were ﬁxed with 10% formaldehyde 24–48 h after transfection, and permealized with 0.5% Triton X-100 then blocked with 5% goat serum.