CCDC134 controls Gp96-dependent maturation and stability of plasma membrane and endolysosomal TLRs. (A) TNFα production of indicated Hoxb8-macrophages stimulated for 24 h with Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (10 ng/ml), R848 (0.1 μg/ml), CpG-B (ODN1668, 1 μM), or flagellin (0.1 μg/ml). Untr.: untreated. (B–D) Immunoblots of indicated Hoxb8-macrophages untreated (B) or stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) for 0–1 h (C) or Pam3CSK4 (0.1 μg/ml), LPS (10 ng/ml), and flagellin (0.1 μg/ml) for 0–1 h (D). (E and F) Immunoblots of indicated Hoxb8-macrophages. short exp.: short exposure; Mat.: mature; Imat.: immature, F: full length; C: cleaved form. (G) Quantification of TLR2, 4, 5, or 7 protein levels in sgRen or sgCCDC134 Hoxb8-macrophages measured by surface (left upper panel) or surface and intracellular (intra.) (left lower panel) staining and quantified by gMFI (relative to respective sgRen). Representative histograms of surface (right upper panel) or intracellular (right lower panel). gMFI surface staining normalized to sgRen TLR2 P value = 0.0053, TLR4 P value = 0.0063, TLR5 P value = 0.0093; gMFI surface and intracellular staining normalized to sgRen TLR2 P value = 0.0002, TLR4 P value = 0.0256, TLR5 P value = ns, TLR7 P value <0.0001, two-tailed one sample t test. (H) Immunoblots of lysate from sgRen or sgCCDC134 Hoxb8-macrophages treated with EndoH (H) or PNGase F (F). Mat.: mature form; Imat.: immature form; dg: deglycosylated. (I) Representative image of sgRen or sgCCDC134 Hoxb8-macrophages stained for TLR7 (scale bar: 5 μm) (left panel) and quantification of TLR7 intensity (right panel). Data are expressed as fold change (FC) and pooled from n = 2. Violins show the variation of individual cells across all experiments; P value <0.0001, two-tailed Mann–Whitney test. (J) Quantification of CD11a, CD18, CD49d, and CD44 protein levels in sgRen or sgCCDC134 Hoxb8-macrophages measured by surface (upper panel) or surface and intracellular (intra.) (bottom panel) staining and quantified by gMFI (relative to respective sgRen). Data show mean ± SD from three independent experiments; gMFI surface staining normalized to sgRen CD11a P value = 0.0316, CD18 P value = 0.0449, CD49d P value = ns, CD44 P value = ns; gMFI surface and intracellular staining normalized to sgRen CD11a P value = 0.0074, CD18 P value = ns, CD49d P value = 0.0029, CD44 P value = ns; two-tailed one sample t test. (K) Volcano plot of quantified proteins in whole proteome of sgCCDC134 versus sgRen Hoxb8-macrophages (upregulated: red, fold change [FC] > 2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01). pink: CCDC134 and Gp96; light pink: Bip and integrins; purple: TLRs; light purple: related to TLRs; green: IFN-stimulated gene proteins (ISGs). In B–F and H, data are representative of two experiments. In A, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In G–J, data show mean ± SD from four or three independent experiments. Source data are available for this figure: SourceData F6.