Rabbit anti myd88

Immunoblot analysis was performed as described earlier [13 (link)], using the following antibodies: rabbit anti-TREM-1 (Abcam, USA), rabbit anti-MyD88 (Cell Signaling, USA), rabbit anti-TRIF (Thermo Fisher Scientific, USA), mouse anti-Syk (Abcam, USA), rabbit anti-p-Syk (Thermo Fisher Scientific, USA), rabbit anti-DAP12 (Cell Signaling, USA), rabbit anti-AKT (Cell Signaling, USA), rabbit anti-p-AKT (Cell Signaling, USA), mouse anti-TLR4 (Santa Cruz, USA), rabbit anti-TLR2 (EMD Millipore, USA), rabbit anti-p38 (Cell Signaling, USA), rabbit anti-p-p38 (Cell Signaling, USA), rabbit anti-ERK (Cell Signaling, USA), rabbit anti-p-ERK (Cell Signaling, USA), rabbit anti-IκB (Cell Signaling, USA), rabbit-anti-p-IκB (Cell Signaling, USA), mouse anti-C/EBP (Santa Cruz, USA), mouse anti-β-actin (Santa Cruz, USA), and rabbit anti-4G10 (Sigma-Aldrich, USA). Either horseradish peroxidase-conjugated goat anti-rabbit antibody or goat anti-mouse antibody (Cell Signaling, USA) was used as a secondary antibody. R406 was purchased from Selleckchem.

EVs were separated from the cells, lysed in IP buffer (cat#87788; Thermo Fisher) with Protease and Phosphatase Inhibitor (Cat#1861281; Thermo Fisher), and agitated for 1 h at 4 °C shaking. The IP buffer consists of 1% NP40, 125 mM NaCl and 25 mM Tris/HCL pH 7.4. Supernatants were transferred to a new reaction tube and a Bradford assay performed. The samples were stored at –80 °C until use. Lysed samples were preincubated with beads for 30 min at 4 °C and centrifuged to collected supernatants. Mouse anti-IL1R1 antibody (Cat# sc-393998; Santa Cruz) was then added to the protein sample. The antibody-protein complex was incubated overnight at 4 °C with agitation. Prewashed beads were added to the sample and incubated for 3 h by shaking at 4 °C. Afterwards, the magnetic beads were separated from the supernatants and transferred to a new tube where they were washed trice. Finally, samples were resuspended in 2 × reducing Laemmli SDS sample buffer. Unbound samples served as a control. After an incubation for 10 min at 70 °C the beads were removed and the supernatants of the elutions were stored at -20 °C. Rabbit anti-pIRAK4 (Cat#11927; Cell signaling Technology®) and rabbit anti-MyD88 (Cat#4283; Cell signaling Technology®) antibodies were used for WB detection.