Anti myd88

The TLR2 ligand Pam₃CSK₄-biotin (Invivogen) was resuspended to a concentration of 1mg/ml in endotoxin-free water. Streptavidin coated 2.8μm magnetic beads (Dynabeads M-270) or streptavidin coated 1μm red (580/605) NeutrAvidin Fluospheres (Invitrogen) were incubated with 1mg/ml of Pam₃CSK₄-biotin for 1hour at 4°C. Beads were spun down and washed in DMEM 10% FBS media twice before resuspending to a concentration of 1×10⁵ beads/μl. The purity of the ligand from contaminants that may activate other receptors was subsequently tested by stimulating TLR2 deficient cells and measuring secretion of cytokines and activation of type I interferons as described below. There was no increase in cytokine or type I interferon expression of TLR2 knockout cells when stimulated with Pam₃CSK₄-biotin.
Immunoblot and confocal microscopy studies were performed using the indicated antibodies: anti-VAMP3 1:1000 (Synaptic Systems), anti-Rab5 1:1000 (Santa Cruz Biotechnology), anti-LAMP1 1:1000 (eBiosciences), anti-Jnk2 1:1000 (Santa Cruz), anti-MyD88 1:1000 (R&D Systems), anti-TICAM2 (TRAM) 1:1000 (Santa Cruz Biotechnology) and anti-phosphoIRF3 1:1000 (Cell Signaling). Specificity of the anti-MyD88 and anti-TRAM antibodies were tested in MyD88 and TRAM deficient cells, respectively.

Cells were plated on 10cm tissue-culture treated dishes and grown to confluency (10⁷ cells/plate overnight). Cells were stimulated with ligand as indicated, then lysed in 700μL of buffer containing 1% NP-40, 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol and protease/phosphatase inhibitors (Roche). 100 μL of cleared lysate was retained for analysis (input) and remaining 600μL was incubated overnight at 4°C with 1μg anti-MyD88 (R&D sciences) or anti-IRAK2 (ProSci). The following day, 50μL of protein G sepharose (GE healthcare) was added for 1 hour. Alternatively, cleared lysates were incubated with Neutravidin agarose beads (Thermo) for 2 hours. Beads were washed 3× with lysis buffer, then proteins were extracted by adding 50μL 2× Laemmli buffer, electrophoresed and immunoblotted with the indicated antibodies using standard conditions. The following antibodies were used: anti-MyD88 (R&D), anti-IRAK2 (Prosci), anti-HA (3F10, Roche), anti-GFP (JL-8, Clontech) and anti-actin (ac-15, Sigma). Anti-IRAK4 was kindly provided by Shizuo Akira.