Specificity of Cav-1 binding to M2 of human Influenza A virus and the participation of cholesterol. A. Pull-down experiments using biotinylated peptides representing the wt M2 CBD or a mutated sequence where aromatic residues in the CBD were changed to alanine. For Cav-1 detection after Western Blot a rabbit polyclonal antibody was used. B. Co-immunoprecipitation (Co-IP) experiments with pM2PR8-EGFP transfected or A/PR8 virus-infected cells. a. Lysates of pEP24c-transfected MDCK cells were processed for co-immumoprecipitation (polyclonal anti-Cav-1 antibody) followed by Western Blot detection of M2 (14C2). b. (Co-IP) of lysates of pM2PR8-EGFP transfected MDCK cells with or without cholesterol depletion by addition of methyl-β-cyclodextran (MβCD) (+) or mock-treatment (-) by rabbit polyclonal anti-Cav-1 antibody (Co-IP) and monoclonal mouse anti-EGFP antibody (indirect M2 detection) were used. MDCK ID: Lysates from transfected cells processed for immunodetection only. c. Lysates of A/PR8 Virus infected cells were processed for immunodetection (MDCK ID) or co-immunoprecipiation (CoIP) after cholesterol depletion with MBCD (+) or mock-treatment (-). Left panel: CoIP using rabbit anti-Cav-1 pAb and detection with anti-M2 antibody (14C2). Right panel: Co-IP: mouse anti-EGFP mAb.Detection: rabbit anti-Cav-1 pAb.MDCK ID: lysates from infected MDCK cells processed for immuno-detection.