Fig. 3. Interference with MIIA expression or function inhibits the ligand-induced endocytosis of CXCR4. (A) Western blot analysis of J77 cells transfected with siRNA oligonucleotides and stained for MIIA, MIIB and tubulin. Numbers depict the quantification of MIIA and MIIB expression, corrected with tubulin loading and related to cells transfected with the negative control oligonucleotide. (B) Flow cytometry analysis of cell surface expression of CXCR4 in cells transfected with siRNA targeting MIIA after 30-minute treatment with 300 nM SDF-1. CXCR4 plasma membrane expression levels in untreated samples were not altered by the transfection of the different siRNA oligonucleotides and were in the range of 90-120 MFI units in the different experiments. Data represent the percentage of CXCR4 with respect to untreated cells (mean± s.d.) of six independent experiments ^*P<0.05. (C) Flow cytometry analysis showing cell surface expression of CXCR4 in cells pretreated for 30 minutes with 50 or 100 µM blebbistatin and then exposed to 300 nM SDF-1 at 37°C for 60 minutes. CXCR4 plasma membrane expression levels before SDF-1 addition were not altered by blebbistatin treatment and ranged 80-100 MFI units in the different experiments. Data represent the percentage of CXCR4 expression with respect to that at 0 minutes in a representative experiment (n=6 experiments). (D) Confocal microscopy analysis of J77 cells transfected with siRNA targeting MIIA (MIIA1) and negative control siRNA. Cells were treated with 300 nM SDF-1 at 37°C for 30 minutes when indicated, and stained for MIIA and clathrin. Bar, 5 µm.