Anti myd88 antibody

For RNA expression analysis, RNA was isolated from HEK293T cells or iPSCs using RNeasy Mini kit (Qiagen), and cDNA generated using QuantiTect Reverse Transcription kit (Qiagen) using primers #2606 5’-aactcatcgagaagaggtgtaggcg and #2607 5’-ccttgtccaaaaccatgatttggtgc. MyD88 amplicons were amplified by PCR using Phire Hot Start II polymerase (Thermo Fisher Scientific). For protein analysis, immunoblotting was performed as previously described (26 (link)) using an anti-MyD88 antibody (Santa Cruz). For loading control, a rabbit anti-β-actin (Cell Signaling Technology) was used.

For immunoprecipitation, whole‐cell lysates were incubated overnight with indicated antibodies plus protein A and G beads (Pierce). For immunoprecipitation with anti‐FLAG, anti‐FLAG agarose gels (BioLegend) were used. Beads were then washed 5 times with low‐salt lysis buffer, and immunoprecipitates were eluted with 4× SDS loading buffer. Immunoblotting was performed by resolving protein lysates on SDS‐PAGE gels, followed by transfer to PVDF membranes (Bio‐Rad) and further incubation of membranes with indicated antibodies overnight. For all blots, EMD Millipore Luminata Western HRP Chemiluminescence substrate was used for protein detection. Anti‐Flag antibody was purchased from Sigma‐Aldrich; anti‐HA antibody was purchased from Roche; anti‐STING, anti‐cGAS, anti‐p‐p38, anti‐p38, anti‐p‐p65, anti‐p‐ERK, anti‐p‐ERK, anti‐JNK, and anti‐p‐JNK antibodies were purchased from Cell Signaling Technology; anti‐MyD88 antibody and anti‐β‐actin antibody were purchased from Santa Cruz Biotechnology.

Co-immunoprecipitation studies were performed as previously described^{5 (link)} using anti-MYD88 antibody (Santa Cruz Biotechnology) and SYK, p-SYK(Y525/Y526) antibodies (Cell Signaling Technologies). Briefly, cells were lysed with Co-IP buffer (Thermo Fisher Scientific) supplemented with 1 mM sodium orthovanadate, 10 mM NaF, 1 × protease inhibitors cocktail for 15 min on ice, and then centrifuged at 2600 × g for 5 min. Supernatants (2 mg total protein) were incubated with 2–4 µg of antibodies at 4 °C for 30 min, followed by incubation with protein A/G-coated magnetic beads (EMD Millipore) for another 30 min at 4 °C. After samples were washed four times with ice-cold lysis buffer on a magnetic stand, proteins were eluted using SDS-PAGE loading buffer for further analysis.