PDI colocalizes and is associated with CD4 and CXCR4 in the presence of gp120. (A) Sup T-1 cells were attached to poly-L-lysine slide and treated in the presence (right panel) or absence of soluble gp120 III_B (left panel). Cells were then fixed in 2% paraformaldehyde and reacted with anti-CD4 rat MAb, anti-PDI rabbit PAb, and anti-CXCR4 mouse MAb. Subsequently, cells were incubated with secondary antirat conjugated with Alexa Fluor 647, antirabbit Cy2, and antimouse Alexa Fluor 555 Abs. Cells were mounted in Fluoromount-G and analyzed by confocal microscopy. PDI is shown as green, CXCR4 is shown as red, and CD4 is shown as blue; white indicates regions of triple, PDI/CD4/CXCR4 colocalization. The data shown are representative of the staining detected in more than 70% of analyzed cells. Original magnification, × 63. (B) HOS-CD4⁺/CXCR4⁺ cells were pretreated with gp120 III_B followed by lysis and coimmunoprecipitation with the anti-CXCR4 MAb, 4G10. Lanes 1 to 3 were probed with anti-PDI PAb, lanes 4 and 5 with an anti-CD4 rabbit serum, and lanes 6 and 7 with an anti-gp120 rabbit serum. A negative control was prepared from HOS-CD4⁺/CXCR4⁺ cell lysate not exposed to 4G10 MAb (lane 1), lane 2 represents PDI coimmunoprecipitated from HOS-CD4⁺/CXCR4⁺ cells in the presence of anti-CXCR4 Ab, and lane 3 represents soluble PDI included as a control for band identity. Lane 4 represents CD4 coimmunoprecipitated from HOS-CD4⁺/CXCR4⁺ cells in the presence of anti-CXCR4 Ab. Lane 5 is a positive CD4 control prepared from HOS-CD4⁺/CXCR4⁺ cell lysate by adding the anti-CD4 MAb, Leu3A (3 μg/5 × 10⁶ cells) and subsequent precipitation with protein G beads. Lane 6 indicates gp120 coimmunoprecipitation from HOS-CD4⁺/CXCR4⁺ cells in the presence of anti-CXCR4 Ab; lane 7 represents soluble gp120 included as a control for band identity.