Journal: The Journal of Biological Chemistry
Article Title: The CIS association of CD47 with integrin Mac-1 regulates macrophage responses by stabilizing the extended integrin conformation
doi: 10.1016/j.jbc.2023.103024
Figure Lengend Snippet: Association of CD47 with Mac-1 on the surface of various Mac-1–expressing cells probed by immunoprecipitation analyses.A, purified WT and CD47-deficient inflammatory peritoneal mouse macrophages were lysed and immunoprecipitated with rabbit polyclonal anti-αM antibody, rabbit polyclonal anti-CD47 antibody, or control rabbit IgG that was used as a specificity control. Blots were analyzed with rabbit polyclonal antibodies against the αM or CD47. The lysates of WT and CD47-deficient macrophages were analyzed by Western blotting using anti-αM and CD47 antibodies. B, murine IC-21 macrophages were lysed and immunoprecipitated with anti-αM rabbit polyclonal antibody or rabbit polyclonal anti-CD47 antibody, and blots were analyzed with rabbit polyclonal antibody against the αM or CD47. C, biotinylated Mac-1-HEK293 cells were lysed and immunoprecipitated with mAb 44a against the αM subunit or isotype control IgG1. Blots were disclosed with streptavidin-conjugated horseradish peroxidase (HRP). The molecular weight of the αM (165 kDa) and β2 (95 kDa) integrin subunits and CD47 (47 kDa) are indicated on the right of the panel. D, suspended (denoted “s”) or adherent (denoted “a”) Mac-1-HEK293 cells were lysed and immunoprecipitated with anti-αM mAb 44a or anti-β2 mAb IB4. Blots were analyzed with anti-αM, anti-β2, and anti-CD47 antibodies. E, the ratios of CD47 to the αM and β2 integrin subunits in the immunoprecipitates from suspended and adherent cells were determined from the densitometry analyses of blots. The ratio of CD47 to each integrin subunit in suspended cells was taken as 1.0. F, lysates of biotinylated Mac-1-HEK293 cells were immunoprecipitated with anti-CD47 mAb B6H12; then immunoprecipitates were subjected to Western blotting probed with streptavidin-HRP (left panel; 1 IP). After the first round of immunoprecipitation, the supernatant was immunoprecipitated with anti-αM mAb 44a (middle panel; 2 IP). The third round of immunoprecipitation (3 IP) was performed using anti-β1 mAb (right panel). M, molecular weight markers.
Article Snippet: The rabbit polyclonal anti-human/mouse CD47 antibody (catalog #CD47-101AP) was from FabGennix.
Techniques: Expressing, Immunoprecipitation, Purification, Western Blot, Molecular Weight
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