DM-induced effects on CXCL12 migration appear to be Lck-, GCR-, and CD45-dependent. (A) Resting T cells were treated with CXCL12 (100 ng/mL), DM (1 μM), or the combination for 5 minutes and were fixed with 2% paraformaldehyde for 1 hour; 10% donkey serum was used for 1 hour at 37°C to block the slides followed by staining with anti-CD45 antibody for 1 hour. The cells were then permeabilized with 90% methanol for 30 minutes at −20°C. Anti-Lck and GC receptor antibodies were added together in blocking buffer, and the slides were incubated overnight at 4°C. Secondary antibody corresponding for each primary antibody labeled with Alexa Fluor 555, Alexa Fluor 647, and Alexa Fluor 488 for the Lck, GR, and CD45, respectively, was added to the slides and incubated for 1 hour at room temperature. The slides were then mounted with Prolong Gold (Invitrogen) mounting media, and image acquisition was done by a confocal microscope (Carl Zeiss) as previously described.^50,51  Isotype controls for each primary antibody were also used in parallel as negative controls. Blue, green, and red indicated the presence of GCR, CD45, and Lck, respectively, and most right panel indicated by the merge of all 3 colors (white). Development of white patches on the merge-image indicated the colocalization of 3 molecules. Single-plane images were examined. (B) Primary human T cells were treated with CXCL12 (100 ng/mL) or DM (1 μM) or a combination for 5 minutes after which the cells were lysed with RIPA buffer. CD45, Lck, and GCR were immunoprecipitated using antibodies to total CD45, Lck, and GCR, respectively. Equivalent amounts of protein were used for immunoprecipitation, and antigen was pulled down from the lysate with antibody-coupled agarose beads after which immunoblotting for each of molecules was performed. Arrows are shown for some of the blots to focus on the bands of interest. See Figure S3 for further quantitation of colocalization after treatment.