Expression of HA-epitope–tagged 4.1R▵E2′,4,5 isoform in transfected COS-7 cells. COS-7 cells were transfected with HA-epitope–tagged 4.1R⁸⁰▵E2′,4,5 isoform (exon map shown in [A]). (B) Protein was immunoprecipitated from precleared cell lysates using a polyclonal anti–HA-epitope tag antibody and analyzed by SDS-PAGE on a 7% acrylamide gel. The HA-epitope–tagged protein migrated at ∼68 kD. Lane 1, cells transfected with empty expression vector; lane 2, cells transfected with HA-tagged 4.1-R⁸⁰▵E2′,4,5. HA-tagged 4.1R isoforms (←). IgG heavy chains migrating ∼55 kD (+). (C) In other experiments, transfected cells were analyzed by immunofluorescence microscopy. Cells were fixed with 3% paraformaldehyde, permeabilized with 0.5% Triton X-100, and double-stained using a monoclonal anti–HA-epitope tag antibody and a polyclonal anti-4.1R antibody raised against the 21 amino acids of exon 16 as primary antibodies and anti-mouse IgG coupled to Texas red and anti-rabbit IgG coupled to FITC as secondary antibodies. Cells, reacting with the HA-epitope tag antibody, showed very strong nuclear expression of 4.1R▵E2′,4,5 with exclusion of the nucleoli and weak cytoplasmic staining (a). An identical pattern of staining was obtained with the anti-4.1R peptide antibody (b). Superimposed images of (a) and (b) are shown in (c). Scale bar: 10μm.