Anti mouse agarose

1 mg of cell lysate was immunoprecipitated with 2 μg anti-ARF3 antibody (610784; BD) overnight at 4°C with gentle rotation. Either anti-mouse agarose or mouse agarose (IgG control; both Sigma-Aldrich) were added for 1 h at 4°C with rotation. Samples were washed three times in RIPA lysis buffer, separated by SDS–PAGE, and immunoblotted. n = 3 experiments from independent lysate preparations and IPs.

Imiquimod (R837) was from InvivoGen. LPS (Escherichia coli type), anti-FLAG, PMA, anti-mouse agarose, anti-HA (clone HA-7) agarose, anti-FLAG (M2) agarose, HA peptide, and 3×FLAG peptide were from Sigma-Aldrich. Monoclonal anti-HA 3F10 and complete protease inhibitor tablets were from Roche. Anti-human CD107A (Lamp1) was from AbD Serotec. Anti-calnexin C-20 was from Santa Cruz Biotechnology, and anti-EEA1 was from Cell Signaling Technology. Anti-rat HRP, anti-mouse HRP, and anti-goat HRP were from R&D Systems, and anti-IL-8–allophycocyanin was from BD Pharmingen. Anti-rabbit Alexa Fluor 555, anti-mouse Alexa Fluor 647, and streptavidin-coated Dynabeads were from Invitrogen. Goat anti-mouse Abberior STAR 440SX-conjugated Ab was a kind gift from Christian Eggeling (University of Oxford). For hIL-8 ELISA, purified anti–hIL-8, monoclonal (clone G265-5; #554716) and biotinylated anti–hIL-8, monoclonal (#554718) were used. The cell surface biotinylation kit was from Pierce Thermo Scientific. The QuikChange II XL Site-Directed Mutagenesis Kit was from Agilent Technologies.

Immune complexes were collected when 1 mg of cell lysate was immunoprecipitated with anti-Met antibody (CST 3127, 1:50) overnight at 4 ^oC with rotation. Anti-mouse agarose or mouse agarose (both Sigma) were added for 1 h at 4 °C prior to three washes in lysis buffer. Samples were then separated by SDS–PAGE, transferred to a PVDF membrane and immunoblotted. Lysates from cells expressing GFP-tagged proteins were immunoprecipitated using a GFP-Trap Kit (Chromotek) as per manufacturer’s instructions and immunoblotting performed as above. n = 3 and quantitation is shown as mean ± s.d.