Intracellular accumulation of the Δ319 G-CSFR. (A) Immunoblot analysis of the G-CSFR in conditioned media (CM) or whole cell lysates (WCLs) fromΔ319 transfectants. CM or lysates were prepared from CHO cells transfected with theΔ319 G-CSFR fused to a Myc-epitope tag. The samples were immunoprecipitated with an anti-Myc antibody and immunoblotted with anti–G-CSFR antibody recognizing aa 25 to 200 of the G-CSFR (sc9173). Samples are from 3 independent Δ319 clones. (Lanes 1-3) CM from the 3 different clones; samples; (lanes 4-6) WCLs from the same samples; Neg indicates untransfected cells as a negative control; Pos, WCLs from transiently transfected CHO cells as a positive control. (B) Subcellular localization of the Δ319 G-CSFR. CHO cells transfected with the Δ716 G-CSFR (left) which accumulates at the cell surface or the Δ319 G-CSFR (right) were grown on glass coverslips for 48 hours, incubated with anti–G-CSFR (sc9173), washed, then stained with Alexa633-conjugated anti-rabbit antibody (red), followed by Hoechst stain for nuclei (blue). Cells were visualized on a Zeiss LSM 510 multiphoton confocal microscope at × 400 amplification.