Phagosome Isolation and Protein Analysis

Detailed descriptions of reagents and experiments can be found in Supplementary Methods. All stable cell lines were generated by retroviral gene transfer. For phagosome isolation, RAW cells were fed latex beads (Polysciences), the cells were disrupted by Dounce homogenization, and latex bead containing phagosomes were purified on a 62%−10% sucrose step gradient in a Beckman SW40Ti centrifuge. After purification, phagosomes were lysed by addition of TritonX-100, and proteins in phagosomes were separated by SDS-PAGE and visualized by immunoblot. Pulse/chase analyses were performed by starving cells of cysteine/methionine for 1 hour, pulsing with ³⁵S-cysteine/methionine for 45 minutes, and chasing in molar excess of unlabeled cysteine/methionine for the indicated time periods. After lysis, radiolabeled proteins were immunoprecipated and visualized by SDS-PAGE. Deglycosylation assays were performed on immunoprecipitated proteins using endoglycosidaseH or PNGaseF (both from NEB) with supplied buffers according to manufacturer's instructions. Proteins were separated by SDS-PAGE and visualized by immunoblot. In vitro proteolysis with recombinant cathepsins (Biomol) was performed according to manufacturer guidelines. For MyD88/TLR9 coimmunoprecipitations, cells were lysed in RIPA Buffer before or after stimulation with CpG, and protein complexes were precipitated with anti-FLAG matrix. For CpG binding assays, TLR9-HA containing lysates were incubated with Biotin-CpG followed by precipitation with streptavidin matrix. In both cases, precipitated proteins were visualized by SDS-PAGE followed by immunoblot. NF-κB luciferase assays were performed by transient transfection of HEK293T cells with an NF-κB luciferase reporter plasmid and the indicated expression plasmids using LTX transfection reagent (Invitrogen) according to manufacturer's instructions. C57/Bl/6 mice were purchased from Jackson Laboratories. All knockout mice have been previously described19 (link)-22 (link). Mice were housed within animal facilities at UC Berkeley or UCSF according to IACUC guidelines.