Figure 2

FLAG and EGFP expression in representative NRP-152 and BPH-1 clones transfected with either pBABE-S3c or pIRES-S3c. NRP-152 and BPH-1 cells were transfected with pBABE-S3c or pIRES-S3c, which bear the FLAG epitope on the S3c gene. Clones were derived by limit dilution, as described in Materials & Methods. Panels A–D: In all histograms, the marker M1 sets the region of positively fluorescent cells for determining the percent positive cells. Panels A & B: Fixed cells were permeabilized and stained with anti-FLAG M1 Ab (Sigma), as described in Materials & Methods. Controls for staining were included, as described. Panel A: Transfected NRP-152 cells. Thin line = 152-S3c; thick line = NRP-152. Panel B: Transfected BPH-1 cells. Thin line = BPH-1; thick line = BPH-S3c. Panels C & D: NRP-152 and BPH-1 cells transfected with pIRES-S3c were analyzed for EGFP fluorescence, following selection. Panel C: Transfected NRP-152 cells. Thin line = NRP-152; thin line = 152-S3c. Panel D: Transfected BPH-1 cells. Thick line = BPH-1; thin line = BPH-S3c. Panel E: Immunoprecipitation followed by Western blot showing EGFP expression in transfected NRP-152 and BPH cells. Note the lack of EGFP bands for parental lines NRP-152 and BPH-1, whereas EGFP was detected using EGFP-specific Ab (Pharmingen) in lanes for 152-S3c and BPH-S3c. Methods: NRP-152, 152-S3c, BPH-1, and BPH-S3c cells were lysed in buffer. Equal amounts of protein in cell lysates were pre-cleared with Protein A/G beads, then precipitated with anti-FLAG AB plus Protein A/G beads with rotation in the cold. The pelleted beads plus proteins were separated on 12% SDS gels, transferred to PVDF membranes, then blotted with Ab to EGFP. Enhanced chemifluorescence was used to reveal the 27 kD bands corresponding to EGFP.