a, b, Validation of the anti-phospho-UNC93B1 polyclonal UNC93B1 antibody. a, Immunoblots demonstrating the specificity of the phospho-specific antibodies generated against Ser547 and Ser550 in the UNC93B1 C-tail. Varying quantities of synthesized peptides corresponding to the UNC93B1 C-terminal regulatory region with (P-UNC93B1-C) and without (NP-UNC93B1-C) phosphorylated Ser547 and Ser550 were dropped onto membrane and probed with rabbit phospho-specific, affinity-purified polyclonal anti-UNC93B1 IgG. Data are representative of two independent experiments. b, Phospho-specific polyclonal antibodies detect both phosphorylated Ser547 and Ser550. UNC93B1 was isolated from UNC93B1-deficient RAW macrophages expressing UNC93B1 mutants S547A, S550A or S547A/S550A by Flag immunoprecipitation followed by immunoblot with phospho-specific polyclonal antibodies. Data are representative of at least three independent experiments. c, Intracellular cytokine staining of TNF in macrophage lines expressing the indicated UNC93B1 alleles and stimulated with CpG (10 nM), R848 (10 ng ml−1), ssRNA (1 µg ml−1), poly(I:C) (20 µg ml−1), or LPS (10 ng ml−1). Grey histograms are unstimulated controls. d, TNF production, measured by ELISA, from the indicated RAW macrophage lines after stimulation for 8 h with LPS (50 ng ml−1). Data are mean ± s.d., n = 3 biological replicates. Data are representative of three independent experiments. e, Levels of phosphorylated p38 and JNK, as measured by immunoblot, in lysates of the indicated RAW macrophage cells stimulated with R848 (50 ng ml−1). Data are representative of two independent experiments.