JCS039057 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Rottlerin treatment indicates that phosphorylation of VASP at Ser239 is crucical for binding to CXCR2. (A) Differentiated HL-60-CXCR2 cells were pretreated with DMSO or 5 µM Rottlerin for 15 minutes, stimulated with 100 ng/ml CXCL8 for 0, 1, and 5 minutes, lysed, then incubated with either normal rabbit IgG- (Mock IgG) or rabbit anti-CXCR2 antibody-coupled Sepharose. Immunoprecipitated proteins were eluted and analyzed by SDS-PAGE and western blot (IB) for VASP Ser239-P and total VASP. Fold changes for total Vasp were quantified using Odyssey 2.1 software (LI-COR Biosciences, Lincoln, NE). The numbers for fold changes for total VASP represent the average intensity of all pixels within the lane profile for a given row of pixels. (B) Lysates from S1(A) were analyzed by western blot analysis using antibodies specific for total VASP.

• Supplemental Figure S2 -

  Fig. S2. H-89 treatment of dHL60 cells shows that phosphorylation of VASP at Ser157 is dispensable for its binding to CXCR2. (A) Differentiated HL-60 cells were incubated with DMSO or 20 µM H-89 for 1 hour at 37°C following which they were stimulated for 0, 1 and 5 minutes with 10 nM CXCL8 at 37°C. The cells were lysed in the co-immunoprecipitation buffer and CXCR2 was immunoprecipitated with rabbit anti-CXCR2 or control rabbit antibody Sepharose. The beads were washed three times with the coimmunoprecipitation buffer and once with without detergent. Bound proteins were eluted with Laemmli sample buffer and any VASP that co-immunoprecipitated with CXCR2 was analyzed by 10% SDS-PAGE and western blot analysis. The blots were blocked in 5% non-fat dry milk in Tris-buffered saline (TBS) and probed with mouse anti-Ser157-P-VASP antibody (Calbiochem, San Diego, CA; 1:500) and rabbit total VASP antibody (Cell Signaling Technology, Inc., Danvers, MA; 1:1000 dilution). Total VASP and Ser157-P was detected with either affinity-purified donkey anti-rabbit secondary antibody tagged with IR dye 800 or with affinity purified goat anti-mouse antibody tagged with Alexa Fluor 680. The protein bands are detected by scanning the wet, drained blot in an Odyssey detection system (LI-COR Biosciences, Lincoln, NE). The images are processed initially using LI-COR odyssey software followed by Photoshop computer program (Adobe Systems, San Jose, CA). The fold total VASP changes in the co-immunoprecipitation experiment were analyzed using Image J software ver 1.41o. (B) Lysates from the co-immunoprecipitation experiment were analyzed for the H-89 mediated inhibition of phosphorylation at Ser157 by PKA.