(a) Peritoneal Mφ from tlr3^−/−, tlr2^−/−tlr4^−/−, hsp90b1 knockout (KO) and control mice were stimulated with polyI:C (100 μg ml⁻¹), lipopolysaccharide (LPS) (1 μg ml⁻¹) or medium (none) in the presence of brefeldin A (BFA) for 6 h. Cells were then stained and analysed for intracellular cytokine by flow cytometry. Numbers represent the percentage of cells in each quadrant over the total number of cells analysed. (b) Purified CD19⁺ splenic-B cells from various mice were stimulated with polyI:C (100 μg ml⁻¹), LPS (1 μg ml⁻¹) or phorbol 12-myristate-13-acetate and ionomycin (P/I) for 6 h. Cells were then analysed for surface CD69 by flow cytometry. Open histograms represent CD69 stain and shaded histograms are isotype controls. (c) pUNO-TLR3HA or pUNO-TLR9HA expression vectors were introduced into WT and gp96 mutant pre-B cells. Cells were stimulated with polyI:C (100 μg ml⁻¹) or LPS (1 μg ml⁻¹) for 12 h, before flow cytometric analysis for nuclear factor-κB-driven green fluorescent protein (GFP). Numbers represent % of GFP⁺ cells. Three experiments were conducted with similar results.

(a) Colocalization of gp96 with CNPY3-FLAG in Raw 264.7 macrophages by immunofluorescence microscopy after costaining with antibody against gp96 and FLAG. Scale bar, 20 μm. (b) gp96 was immunoprecipitated (IP) from Raw 264.7 macrophages or bone marrow-derived macrophages (BMDMs), followed by immunoblot (IB) for gp96 and endogenous CNPY3. (c) HEK293 cells were mock transfected or transfected with CNPY3-FLAG. After 48 h, cells were immunoprecipitated with FLAG or gp96 antibody, resolved on SDS–PAGE and immunoblotted for gp96 and FLAG. As a control, whole-cell lysates (WCLs) were also blotted for CNPY3 and gp96. (d) WT pre-B cells were immunoprecipitated for CNPY3-FLAG or gp96, or with isotype (ISO) control antibody, followed by IB for CNPY3 and gp96. (e) IP of N-fragment of gp96 (N355) or C-fragment of gp96 (C150) from mutant pre-B cells, followed by IB for CNPY3-FLAG and gp96. (f) Sequential IP analysis for TLR9–gp96–CNPY3 interaction in pre-B and HEK293 cells. IP of TLR9-HA, followed by elution with HA peptide, re-IP with FLAG antibody for CNPY3-FLAG and IB for indicated molecules. (g) IP of TLR9HA or TLR3HA from pre-B cells, followed by elution with HA peptide, gel filtration on Superdex 200 column and IB identification of proteins in indicated fractions. (h) IP of gp96 or gp96^E103A mutant from pre-B cells expressing the respective proteins, followed by IB for CNPY3-FLAG and gp96. (i) gp96-null pre-B cell lines were spin-infected with MigR1-gp96^E103A or MigR1-gp96^WT retroviral vectors, followed by staining of various gp96 client proteins on cell surface and flow cytometric analysis. (j) Raw 264.7 cells were transfected with WT CNPY3 or mutant CNPY3 (M145K). IP of gp96 was performed, followed by IB for CNPY3-FLAG or gp96. Results were confirmed by multiple experiments.

(a) Characterization of gp96, recombinant GST and CNPY3-GST by immunoblot and coomassie blue staining after resolution on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). (b) GST or CNPY3-GST was preincubated with glutathione beads, followed sequentially by blocking with bovine serum albumin (BSA) and incubation with gp96 for 2 h. The pulldown materials were immunoblotted for gp96 or GST as indicated. (c) Plate-bound GST or CNPT3-GST was incubated with gp96, followed by washing, blocking with BSA and detection of bound gp96 with ELISA in duplicates. Data are means of five independent experiments, error bars indicate standard error of mean. *P0.05. (d) and (e) Effect of adenosine nucleotide on gp96-CNPY3 interaction. Same as in (c), except that various inhibitors at the titrated dose (d) or 1 mM (e) were present during incubation with gp96 (10 μg ml⁻¹). Data points are means of three individual experiments. Assays were performed in duplicates. Error bars indicate standard error of mean. *P<0.05.

(a) CNPY3 mRNA level (mean of duplicates) by a quantitative-PCR (Q-PCR) analysis after CNPY3 short-hairpin RNA (shRNA) interference. (b) Surface TLR4 expression (open histogram) on Raw264.7 cells after shRNA knockdown of CNPY3. Shaded histograms represent staining with isotype control antibody. (c) CNPY3 or control shRNA lentivirus-transduced Raw264.7 cells were stimulated with various TLR ligands for 3 h (Pam3 100 ng ml⁻¹; lipopolysaccharide 200 ng ml⁻¹, R848 50 ng ml⁻¹), followed by measurement of tumour-necrosis factor-α in the supernatant by ELISA. All assays were performed in duplicates. Data are means of three independent experiments, error bar indicates standard error of mean. *P<0.05. (d) Same as (c), except that cells were stimulated with CpG (1 μM) for indicated times. Data points are means of three independent experiments. (e) Total cell lysates were incubated at 37°C for indicated time and blotted for TLR9HA and gp96 in cells transduced with CNPY3 (S) or control (C) shRNA lentiviral vector. (f) gp96 IP was performed using cells in (e) (top panel), or cells pretreated with CNPY3 siRNA (bottom panel), followed by IB for TLR9HA and gp96. Protein expression in whole-cell lysate is also shown.

(a) Expression of endogenous CNPY3 and gp96 by Raw264.7 after transduction with empty vector (EV), and one or two rounds of gp96 shRNA lentivector. β-Actin is shown as a loading control. (b) Lack of cleaved form of TLR9 (TLR9 m) in the absence of gp96 or CNPY3. Total lysates of wild-type (WT) Raw264.7, cells that were knocked down (KD) for gp96 (gp96 KD) or CNPY3 (CNPY3 KD) were untreated, or treated with Endo H or PNGase F, followed by IB for TLR9HA. (c) WT or gp96 Mut pre-B cells were immunoblotted for TLR9-HA, gp96, CNPY3-FLAG and β-actin. (d) IP of CNPY3 from WT or gp96 KO cells, followed by IB for indicated proteins. (e) IP of TLR9-HA from WT or gp96 KO cells, followed by IB for indicated proteins. ISO indicated IP with isotype control antibody. Two experiments were conducted with similar results.

(a) Schematic diagram of TLR fusion proteins. HA represents haemagglutinin epitope from the influenza virus. The transmembrane domain of the platelet-derived growth factor (PDGF) receptor is indicated as tm. Signal peptide is not depicted. (b) TLR fusion proteins are detected by HA immunoblotting from retroviral transduced pre-B cells (w-WT, m-Mut cells). TLR3 doublets represent difference in N-linked glycosylation. β-actin is probed as a loading control. (c) WT and gp96 mutant (mut) pre-B cells were transduced with indicated retrovirus, followed by flow cytometric analysis of surface TLR with HA antibody (open histogram). Shaded histograms are staining controls with isotype-matched antibody. Multiple experiments were conducted with similar results. (d) Empty vector (EV) and CNPY3-silenced Raw264.7 cells were transduced with various TLRtm vectors, followed by cell surface staining of TLRtms (open histogram). Shaded histogram represents staining with untransduced cells.

(a) IB of endogenous CNPY3 and gp96 from THP1 and HEK293 cells. (b) HEK293 cells were transfected with TLR9tm or empty vector (EV). After 48 h, TLR9HA from the total cell lysates was immunoprecipitated with HA antibody. The eluate was resolved on SDS–PAGE and stained with Coomassie blue. Individual bands indicated by arrows were cut, trypsin-digested and sequenced by mass spectrometry. The identities of p40-p200 are indicated. The asterisk represents Ig heavy chain. (c) HEK293 cells were transfected transiently with empty vector (EV), TLR9tm (9tm) or TLR3tm (3tm) expression vector, followed by IB, for indicated proteins. (d) HEK293 cells were transfected with TLR9tm or a combination of TLR9tm and CNPY3 expression vector, followed by IB for indicated proteins.

The initial folding and glycosylation of nascent TLR9p0 (TLR9 precursor 0) are mediated by the ER general glycoprotein-folding machinery, including calnexin (CNX) and calreticulin (CRT). CNPY3 and gp96 must form a complex to more efficiently interact with TLR9p1 (TLR9 precursor 1). gp96–CNPY3 accelerates the tertiary folding of TLR9. Folded TLR9p2 (TLR9 precursor 2) then dissociates from the gp96–CNPY3 complex and translocates to the endolysosome with the aid of the membrane protein unc93b. TLR9 completes its maturation cycle after proteolytic cleavage of the first N-terminal 14 LRRs in the endolysosome. Dotted line represents steps that are not fully characterized.