Endogenous NCU-G1 is a nuclear protein. Panel A: S2 cells were transfected with expression vectors for hNCU-G1 (pAc-NCU-G1) or its truncated version pAc-hNCU-G1(Δ exon 6). Whole cell extracts (20 μg/lane) were analyzed by Western blotting using preimmune serum (lanes 1 to 3) or an antiserum raised against a C-terminal 15 amino acid peptide (lanes 5 to 7). Lane 1 and 5: control, untreated S2 cells, lane 2 and 6: S2 cells expressing hNCU-G1, lane 3 and 7: S2 cells expressing hNCU-G1(Δ exon6). Panel B: Cytoplasmic and nuclear extracts (20 μg/lane) from JEG3, RPE and 293 cells were size-fractionated on 10% SDS-PAGE gels and analyzed by Western blotting using NCU-G1 antiserum. Lane 1: JEG3 cytosol, lane 2: JEG3 nuclear extract, lane 3: RPE cytosol, lane 4: RPE nuclear extract, lane 5: 293 cytosol, lane 6: 293 nuclear extract. Panel C: The membranes used in panel B were stripped and analyzed with an antibody against Sp1.