goat polyclonal il 22bp antibody  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2 , and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH . (B) Effect of different maturation stimuli (CpG and LPS + IFN-γ or Poly I:C) on IL22RA2, IL12B , and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB . Extent of maturation is represented as percentage of CD83 + fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB was measured by RT-qPCR using pre-designed SYBR Green primers from the same cells. Representative experiment out of three performed. All primers used are listed in Supplementary Table .

Article Snippet: High binding ELISA plates (Sarstedt, 82.1581.200) were coated with 100 μl of 0.5 μg/ml antigen-purified goat polyclonal IL-22BP antibody (R&D systems, AF1087; immunogen used was recombinant human IL-22BP Thr22-Pro231 coinciding with mature IL-22BPi2) in coating solution (50 mM Tris/HCl, 150 mM NaCl, pH 8,5) and incubated o/n at 4°C.

Techniques: Derivative Assay, Binding Assay, Variant Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, SYBR Green Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Differential secretion, intracellular retention and degradation by the proteasome of IL-22BP isoforms. Data refer to HEK293 cells transiently transfected for 24 h unless otherwise specified. (A) HEK293 cells were transiently transfected with expression vectors encoding IL-22BPi1, BPi2, or BPi3. Intracellular and secreted IL-22BP protein levels were measured by WB (FLAG Ab) and ELISA, respectively. Transfection efficiency was measured by IL22RA2 RT-qPCR relative to the housekeeping gene GAPDH (mean ± SEM; n = 3). (B) Confocal microscopy of IL-22BP isoforms (green, IL-22BP Ab 4 with either ERp72 or trans-Golgi marker TGOLN2 (red) in transfected HEK293 cells; cells were counterstained with DAPI. (C) Secreted recombinant IL-22BPi2 is not detected by WB in unconcentrated conditioned media (CM). Various cytokine expression vectors, as indicated, were transiently transfected and CM were collected and immunoblotted for FLAG. (D) Detection of IL-22BP isoforms in cell lysates (CL) and acetone-precipitated CM (AP) by FLAG Ab. Secreted IL-22BPi2 was treated with PNGase F or Endo H. (E) Intracelullar isoforms of IL-22BP were treated with PNGase F or Endo H and detected by FLAG Ab. (F) HEK293 cells were transfected for 24 h with expression vectors for either IL-2 or IL-2EX4. The latter was generated by subcloning exon-4 from IL22RA2v1 into the open reading frame of IL-2 through its unique Xba1 position. Detection of resulting proteins in AP, CL, CM by FLAG Ab. (G) HEK293 cells were transiently transfected with the same expression plasmids as in (A) or IL-2EX4; 24 h later, cells were treated with a mix of proteasome inhibitors (PI) (5 μM lactacystin, 5 μM MG132, and 1 mM epoxomicin), and 18 h later cells were lysed and immunoblotted for FLAG using GAPDH as a loading control. All data are from at least 3 independent experiments.

Article Snippet: High binding ELISA plates (Sarstedt, 82.1581.200) were coated with 100 μl of 0.5 μg/ml antigen-purified goat polyclonal IL-22BP antibody (R&D systems, AF1087; immunogen used was recombinant human IL-22BP Thr22-Pro231 coinciding with mature IL-22BPi2) in coating solution (50 mM Tris/HCl, 150 mM NaCl, pH 8,5) and incubated o/n at 4°C.

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Confocal Microscopy, Marker, Recombinant, Generated, Subcloning

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BPi1 does not interact with IL-22 or IL-22BPi2. (A) A549 cells were exposed for 30 minutes to dilutions of IL-22-containing culture medium (CM) previously produced by IL22 -transfected HEK293 cells. A549 cell lysates (CL) were immunoblotted for pSTAT3 and tubulin as loading control. The relative densitometry of pSTAT3 normalized to that of tubulin is also represented. (B) A549 cells were treated with the optimum IL-22 dilution from A (1/512) for different periods of time, lysed and immunoblotted for pSTAT3. An unspecific protein band (u.p.) was used as loading control. (C) IL-22BP concentration in conditioned medium (CM) of transfected HEK293 cells was measured by ELISA, and the indicated amounts in nanograms (ng) of IL-22BPi1 or IL-22BPi2 were pre-incubated for 1 h at 37°C with the selected IL-22 concentration from (A) . A549 were exposed to the pre-incubated combinations for 20 min. An excess of IL-22BPi2 was used as phosphorylation blocking control (lane 3). Cells were lysed and immunoblotted for pSTAT3 and actin as loading control. (D) HeLa cells were co-transfected with the indicated expression plasmids, 24 h later cells were lysed and the CM was subjected to acetone precipitation (AP), and proteins were immunoblotted for FLAG. Intracellular IL-22BPi1 is indicated with dark purple arrows, intracellular and secreted IL-22BPi2 is indicated with green arrows, and co-expressed IL-22 and IL-17 are also indicated. (E) Conditioned media (CM) from 3 independent experiments, in which expression vectors for the three IL-22BP isoforms were individually transfected into HEK293 cells together with either IL-17 or IL-22 expression vectors or an empty vector control (EV), were analyzed 24 h after transfection by ELISA for IL-22BP (mean ± SEM; n = 3; ** p < 0.01 by unpaired t -test). (F) IL-22BPi1 does not interact with IL-22BPi2. IL-22BPi1-MF expression plasmid containing Myc and FLAG tags was co-transfected with an inducible pTRE3G-based vector expressing IL-22BPi2 with only a FLAG tag. After 24 h, cells were induced for IL-22BPi2 production by adding Tet-Express activator to the medium for a further 24 h. Cells were lysed and immunoprecipitated with anti-Myc agarose, the flow-through fractions were then further subjected to FLAG immunoprecipitation. CL and eluted fractions were immunoblotted for FLAG.

Article Snippet: High binding ELISA plates (Sarstedt, 82.1581.200) were coated with 100 μl of 0.5 μg/ml antigen-purified goat polyclonal IL-22BP antibody (R&D systems, AF1087; immunogen used was recombinant human IL-22BP Thr22-Pro231 coinciding with mature IL-22BPi2) in coating solution (50 mM Tris/HCl, 150 mM NaCl, pH 8,5) and incubated o/n at 4°C.

Techniques: Produced, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Blocking Assay, Expressing, Plasmid Preparation, FLAG-tag, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BPi1 and IL-22BPi2, but not IL-22BPi3, are bona fide client proteins of GRP78. ( A,B) Identification of GRP78, GRP94, GRP170 and calnexin in interactomes of IL-22BPi1 and IL-22BPi2. Silver-stained (S.S) gels of proteins (co-)immunoprecipitated (IP) with FLAG resins from HEK293 or HeLa cells transiently transfected with the indicated expression plasmids or control empty vector (EV) are shown. Numbered arrow-heads indicate proteins identified by mass spectrometry included in the inset summary Table (detailed information in Supplementary Data ). Co-immunoprecipitated proteins without DSP treatment were immunoblotted for detection by FLAG Ab. Asterisk, IL-22BPi1; empty arrowhead, IL-22; solid rhombus, IL-22BPi2; empty rhombus, IL-2EX. (Inset) Summary table with the mass spectrometry results from protein identification. # Unique peptides, # Peptides, and # PSMs are: the number of peptide sequences unique to a protein group, the number of distinct peptide sequences in the protein group and the total number of identified peptide sequences (peptide spectrum matches) for the protein. (C) IL-22BPi1, IL-22BPi2 and IL-2EX4 bind the substrate-binding domain of GRP78. HEK293 cells were transiently transfected with the indicated constructs and 24 h later were either treated with 100 ng/ml Mut SubAB or SubAB for 3 h. Cell lysates (CL) were co-immunoprecipitated with FLAG agarose. CL and IP proteins were immunoblotted for FLAG or GRP78. (D) HEK293 cells were individually transfected with expression plasmids for the three IL-22BP isoforms together with either GRP78 or GRP78 T37G expression vectors or empty vector (EV). 24 h later secreted protein was measured by ELISA for IL-22BP (mean ± SEM; n = 3; * p < 0.05, ** p < 0.01 by unpaired t -test).

Article Snippet: High binding ELISA plates (Sarstedt, 82.1581.200) were coated with 100 μl of 0.5 μg/ml antigen-purified goat polyclonal IL-22BP antibody (R&D systems, AF1087; immunogen used was recombinant human IL-22BP Thr22-Pro231 coinciding with mature IL-22BPi2) in coating solution (50 mM Tris/HCl, 150 mM NaCl, pH 8,5) and incubated o/n at 4°C.

Techniques: Staining, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Mass Spectrometry, Binding Assay, Construct, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Both IL-22BPi1 and IL-22BPi2 interact with the middle domain of GRP94. (A) HEK293 cells were transiently co-transfected with the indicated expression plasmids, 24 h later cell lysates were immunoprecipitated with FLAG resin to IP IL-22BP isoforms or IL-2EX4, or with S-tag agarose to IP GRP94. (Co-)immunoprecipitated proteins (IP) were immunoblotted for GRP94 and FLAG. (B) A schematic diagram of full-length (WT) and structural mutants of GRP94 used. (C) (Western blot images) HEK293 cells were transiently co-transfected with the indicated expression plasmids, 24 h later cell lysates (CL) were co-immunoprecipitated with Myc agarose. Membranes with CL and IP proteins were sequentially exposed for detection by KDEL and mouse (Ms) or rabbit (Rb) FLAG Abs, or vice versa. MD-GRP94 or ΔA-GRP94 co-immunoprecipitated with IL-22BPi1 or IL-22BPi2 are indicated with black arrows. (Lower) Expression vectors for IL-22BP isoform-1 and−2 were individually transfected into HEK293 cells together with either GRP94 wild-type (WT) or GRP94 mutant vectors (CTD, MD, NTD, or deltaA) or empty vector (EV). Twenty-four hour after transfection, secreted IL-22BP was quantified by ELISA, analyzed by paired t -test on original data and represented as fold change relative to EV condition (mean ± SEM; n = 3; * p < 0.05 by paired t -test, original paired data are represented in Supplementary Figure ).

Article Snippet: High binding ELISA plates (Sarstedt, 82.1581.200) were coated with 100 μl of 0.5 μg/ml antigen-purified goat polyclonal IL-22BP antibody (R&D systems, AF1087; immunogen used was recombinant human IL-22BP Thr22-Pro231 coinciding with mature IL-22BPi2) in coating solution (50 mM Tris/HCl, 150 mM NaCl, pH 8,5) and incubated o/n at 4°C.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: GRP94 ATPase activity is important for IL-22BPi1 and IL-22BPi2 secretion. GRP94 co-localizes with IL-22BPi1 and IL-22BPi2 but not IL-22BPi3. (Left) Confocal microscopy of HEK293 cells co-transfected with IL-22BP isoforms with the indicated GFP-GRP94 fusion constructs (depicted lower right); IL-22BP was stained using an anti-IL-22BP antibody (red, IL-22BP Ab 4), IL-2EX4 was stained with mouse anti-FLAG Ab, and the nucleus was stained using DAPI (blue). Secreted IL-22BP was measured by ELISA in the conditioned media (CM) of HEK293 cells transfected with individual expression vectors for IL-22BP isoforms together with the indicated GRP94 vectors (upper right) (mean ± SEM; n = 4; ** p < 0.01 by unpaired t -test).

Article Snippet: High binding ELISA plates (Sarstedt, 82.1581.200) were coated with 100 μl of 0.5 μg/ml antigen-purified goat polyclonal IL-22BP antibody (R&D systems, AF1087; immunogen used was recombinant human IL-22BP Thr22-Pro231 coinciding with mature IL-22BPi2) in coating solution (50 mM Tris/HCl, 150 mM NaCl, pH 8,5) and incubated o/n at 4°C.

Techniques: Activity Assay, Confocal Microscopy, Transfection, Construct, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BPi1 and IL-2EX4 induce unfolded protein response (UPR) genes. HEK293 cells were transiently transfected with IL-22BPi1, IL-22BPi2, IL-22BPi3, IL-2, IL-2EX4 or empty vector (EV) as control. Cells were collected at the indicated hours after transfection. (A) Expression of different genes related to ER function or UPR were analyzed by RT-qPCR. Each gene expression value is represented as fold change relative to the same time-point expression value of the EV condition and relative to the housekeeping gene GAPDH . Mean ± SEM of three independent experiments. All primers are listed in Supplementary Table . (B) GRP78 and GRP94 protein levels correlate with mRNA levels observed in (A) . Cell lysates (CL) were immunoblotted for FLAG, GRP94, KDEL and tubulin as loading control. (C) IL-22BPi1 and IL-2EX4 cause XPB1 splicing. XBP1 splicing was detected with conventional PCR for the indicated conditions and times. Un-spliced and spliced XBP1 are indicated as XBP1-u or XBP-1s respectively. (D) IL-22BPi2 secretion was not increased when co-expressed with different ratios of IL-22BPi1. HEK293 cells were co-expressed with different ratios of EV:IL-22BPi1:IL-22BPi2 expression plasmids. 48 h later, secreted IL-22BP in conditioned media (CM) was quantified by ELISA (mean ± SEM; n = 3). (E) Cell viability measured with alamarBlue was not compromised by any of the conditions in two different cell lines. Reduction of alamarBlue was measured after 48 h of transfection and assayed for the indicated times and cell lines. Values are represented as percentage of reduction in each condition relative to EV (mean ± SEM; n = 3).

Article Snippet: High binding ELISA plates (Sarstedt, 82.1581.200) were coated with 100 μl of 0.5 μg/ml antigen-purified goat polyclonal IL-22BP antibody (R&D systems, AF1087; immunogen used was recombinant human IL-22BP Thr22-Pro231 coinciding with mature IL-22BPi2) in coating solution (50 mM Tris/HCl, 150 mM NaCl, pH 8,5) and incubated o/n at 4°C.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Silencing of IL22RA2 in immature moDCs reduces GRP78 expression. (A) CD14 + monocytes were stained with DAPI (blue), trans-GOLGI (red) and IL-22BP (green, IL-22BP Ab 4). (B) Overall (best coverage, BC) and isoform-specific IL22RA2 expression levels in classical, intermediate and non-classical monocyte subpopulations (top) from four healthy donors (average ± SEM). Percentages of IL-22BP + monocytes found in each subpopulation by immunofluorescence microscopy (bottom) (mean ± SEM; n = 4; * p < 0.05, Kruskal-Wallis test for comparison of differences in IL22RA2 mRNA levels or IL-22BP + counts in the three subpopulations). (C) IL22RA2 expression levels in classical and intermediate immature moDCs at day 6 of cultivation. moDCs derived from non-classical monocytes showed high levels of cell death and had not experienced any changes in the shape of the cells. High Ct values of HK gene HPRT1 in qPCR compromised correct interpretation of IL22RA2 levels in this subgroup, which was therefore excluded from the graph. (D) Fold change relative to non-targeting siRNA control of IL22RA2 expression in CD14 + monocyte.-derived immature DCs on day 5 of cultivation following IL22RA2 silencing with Viromer Green at two concentrations of siRNA. (E) Silencing as described in (D) was not variant specific and both IL22RA2v1 and v2 were silenced to similar extent. (F) Scheme comparing efficiencies of four distinct silencing strategies on suppression of IL22RA2 mRNA (qPCR) and intracellular IL-22BP (ELISA) in immature CD14 + moDCs using 100 nM IL22RA2 siRNA or non-targeting siRNA in combination with Viromer Green. S, silencing, H, harvest. Bar diagrams represent fold change relative to non-targeting siRNA. (G) Effect of IL22RA2 silencing on GRP78 and ERp44 mRNA levels in CD14 + monocyte-derived immature DCs from healthy donors using silencing strategy B in (F) . CD14 + monocytes were cultivated in DM medium in the absence or presence of AM580 starting on day 0 up to the day of harvest. Average of fold change (±SEM) relative to non-silencing control over 6 (control) and 5 (AM580) independent measurements relative to the housekeeping gene HPRT1. * p < 0.03 by Wilcoxon test.

Article Snippet: High binding ELISA plates (Sarstedt, 82.1581.200) were coated with 100 μl of 0.5 μg/ml antigen-purified goat polyclonal IL-22BP antibody (R&D systems, AF1087; immunogen used was recombinant human IL-22BP Thr22-Pro231 coinciding with mature IL-22BPi2) in coating solution (50 mM Tris/HCl, 150 mM NaCl, pH 8,5) and incubated o/n at 4°C.

Techniques: Expressing, Staining, Immunofluorescence, Microscopy, Comparison, Derivative Assay, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2 , and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH . (B) Effect of different maturation stimuli (CpG and LPS + IFN-γ or Poly I:C) on IL22RA2, IL12B , and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB . Extent of maturation is represented as percentage of CD83 + fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB was measured by RT-qPCR using pre-designed SYBR Green primers from the same cells. Representative experiment out of three performed. All primers used are listed in Supplementary Table .

Article Snippet: Antigen-purified goat polyclonal IL-22BP biotinylated antibody (R&D Systems, BAF1087) was added to each well in assay buffer at 0.5 μg/ml dilution and incubated for 1 h at 37°C followed by six washings.

Techniques: Derivative Assay, Binding Assay, Variant Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, SYBR Green Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Differential secretion, intracellular retention and degradation by the proteasome of IL-22BP isoforms. Data refer to HEK293 cells transiently transfected for 24 h unless otherwise specified. (A) HEK293 cells were transiently transfected with expression vectors encoding IL-22BPi1, BPi2, or BPi3. Intracellular and secreted IL-22BP protein levels were measured by WB (FLAG Ab) and ELISA, respectively. Transfection efficiency was measured by IL22RA2 RT-qPCR relative to the housekeeping gene GAPDH (mean ± SEM; n = 3). (B) Confocal microscopy of IL-22BP isoforms (green, IL-22BP Ab 4 with either ERp72 or trans-Golgi marker TGOLN2 (red) in transfected HEK293 cells; cells were counterstained with DAPI. (C) Secreted recombinant IL-22BPi2 is not detected by WB in unconcentrated conditioned media (CM). Various cytokine expression vectors, as indicated, were transiently transfected and CM were collected and immunoblotted for FLAG. (D) Detection of IL-22BP isoforms in cell lysates (CL) and acetone-precipitated CM (AP) by FLAG Ab. Secreted IL-22BPi2 was treated with PNGase F or Endo H. (E) Intracelullar isoforms of IL-22BP were treated with PNGase F or Endo H and detected by FLAG Ab. (F) HEK293 cells were transfected for 24 h with expression vectors for either IL-2 or IL-2EX4. The latter was generated by subcloning exon-4 from IL22RA2v1 into the open reading frame of IL-2 through its unique Xba1 position. Detection of resulting proteins in AP, CL, CM by FLAG Ab. (G) HEK293 cells were transiently transfected with the same expression plasmids as in (A) or IL-2EX4; 24 h later, cells were treated with a mix of proteasome inhibitors (PI) (5 μM lactacystin, 5 μM MG132, and 1 mM epoxomicin), and 18 h later cells were lysed and immunoblotted for FLAG using GAPDH as a loading control. All data are from at least 3 independent experiments.

Article Snippet: Antigen-purified goat polyclonal IL-22BP biotinylated antibody (R&D Systems, BAF1087) was added to each well in assay buffer at 0.5 μg/ml dilution and incubated for 1 h at 37°C followed by six washings.

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Confocal Microscopy, Marker, Recombinant, Generated, Subcloning

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BPi1 does not interact with IL-22 or IL-22BPi2. (A) A549 cells were exposed for 30 minutes to dilutions of IL-22-containing culture medium (CM) previously produced by IL22 -transfected HEK293 cells. A549 cell lysates (CL) were immunoblotted for pSTAT3 and tubulin as loading control. The relative densitometry of pSTAT3 normalized to that of tubulin is also represented. (B) A549 cells were treated with the optimum IL-22 dilution from A (1/512) for different periods of time, lysed and immunoblotted for pSTAT3. An unspecific protein band (u.p.) was used as loading control. (C) IL-22BP concentration in conditioned medium (CM) of transfected HEK293 cells was measured by ELISA, and the indicated amounts in nanograms (ng) of IL-22BPi1 or IL-22BPi2 were pre-incubated for 1 h at 37°C with the selected IL-22 concentration from (A) . A549 were exposed to the pre-incubated combinations for 20 min. An excess of IL-22BPi2 was used as phosphorylation blocking control (lane 3). Cells were lysed and immunoblotted for pSTAT3 and actin as loading control. (D) HeLa cells were co-transfected with the indicated expression plasmids, 24 h later cells were lysed and the CM was subjected to acetone precipitation (AP), and proteins were immunoblotted for FLAG. Intracellular IL-22BPi1 is indicated with dark purple arrows, intracellular and secreted IL-22BPi2 is indicated with green arrows, and co-expressed IL-22 and IL-17 are also indicated. (E) Conditioned media (CM) from 3 independent experiments, in which expression vectors for the three IL-22BP isoforms were individually transfected into HEK293 cells together with either IL-17 or IL-22 expression vectors or an empty vector control (EV), were analyzed 24 h after transfection by ELISA for IL-22BP (mean ± SEM; n = 3; ** p < 0.01 by unpaired t -test). (F) IL-22BPi1 does not interact with IL-22BPi2. IL-22BPi1-MF expression plasmid containing Myc and FLAG tags was co-transfected with an inducible pTRE3G-based vector expressing IL-22BPi2 with only a FLAG tag. After 24 h, cells were induced for IL-22BPi2 production by adding Tet-Express activator to the medium for a further 24 h. Cells were lysed and immunoprecipitated with anti-Myc agarose, the flow-through fractions were then further subjected to FLAG immunoprecipitation. CL and eluted fractions were immunoblotted for FLAG.

Article Snippet: Antigen-purified goat polyclonal IL-22BP biotinylated antibody (R&D Systems, BAF1087) was added to each well in assay buffer at 0.5 μg/ml dilution and incubated for 1 h at 37°C followed by six washings.

Techniques: Produced, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Blocking Assay, Expressing, Plasmid Preparation, FLAG-tag, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BPi1 and IL-22BPi2, but not IL-22BPi3, are bona fide client proteins of GRP78. ( A,B) Identification of GRP78, GRP94, GRP170 and calnexin in interactomes of IL-22BPi1 and IL-22BPi2. Silver-stained (S.S) gels of proteins (co-)immunoprecipitated (IP) with FLAG resins from HEK293 or HeLa cells transiently transfected with the indicated expression plasmids or control empty vector (EV) are shown. Numbered arrow-heads indicate proteins identified by mass spectrometry included in the inset summary Table (detailed information in Supplementary Data ). Co-immunoprecipitated proteins without DSP treatment were immunoblotted for detection by FLAG Ab. Asterisk, IL-22BPi1; empty arrowhead, IL-22; solid rhombus, IL-22BPi2; empty rhombus, IL-2EX. (Inset) Summary table with the mass spectrometry results from protein identification. # Unique peptides, # Peptides, and # PSMs are: the number of peptide sequences unique to a protein group, the number of distinct peptide sequences in the protein group and the total number of identified peptide sequences (peptide spectrum matches) for the protein. (C) IL-22BPi1, IL-22BPi2 and IL-2EX4 bind the substrate-binding domain of GRP78. HEK293 cells were transiently transfected with the indicated constructs and 24 h later were either treated with 100 ng/ml Mut SubAB or SubAB for 3 h. Cell lysates (CL) were co-immunoprecipitated with FLAG agarose. CL and IP proteins were immunoblotted for FLAG or GRP78. (D) HEK293 cells were individually transfected with expression plasmids for the three IL-22BP isoforms together with either GRP78 or GRP78 T37G expression vectors or empty vector (EV). 24 h later secreted protein was measured by ELISA for IL-22BP (mean ± SEM; n = 3; * p < 0.05, ** p < 0.01 by unpaired t -test).

Article Snippet: Antigen-purified goat polyclonal IL-22BP biotinylated antibody (R&D Systems, BAF1087) was added to each well in assay buffer at 0.5 μg/ml dilution and incubated for 1 h at 37°C followed by six washings.

Techniques: Staining, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Mass Spectrometry, Binding Assay, Construct, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Both IL-22BPi1 and IL-22BPi2 interact with the middle domain of GRP94. (A) HEK293 cells were transiently co-transfected with the indicated expression plasmids, 24 h later cell lysates were immunoprecipitated with FLAG resin to IP IL-22BP isoforms or IL-2EX4, or with S-tag agarose to IP GRP94. (Co-)immunoprecipitated proteins (IP) were immunoblotted for GRP94 and FLAG. (B) A schematic diagram of full-length (WT) and structural mutants of GRP94 used. (C) (Western blot images) HEK293 cells were transiently co-transfected with the indicated expression plasmids, 24 h later cell lysates (CL) were co-immunoprecipitated with Myc agarose. Membranes with CL and IP proteins were sequentially exposed for detection by KDEL and mouse (Ms) or rabbit (Rb) FLAG Abs, or vice versa. MD-GRP94 or ΔA-GRP94 co-immunoprecipitated with IL-22BPi1 or IL-22BPi2 are indicated with black arrows. (Lower) Expression vectors for IL-22BP isoform-1 and−2 were individually transfected into HEK293 cells together with either GRP94 wild-type (WT) or GRP94 mutant vectors (CTD, MD, NTD, or deltaA) or empty vector (EV). Twenty-four hour after transfection, secreted IL-22BP was quantified by ELISA, analyzed by paired t -test on original data and represented as fold change relative to EV condition (mean ± SEM; n = 3; * p < 0.05 by paired t -test, original paired data are represented in Supplementary Figure ).

Article Snippet: Antigen-purified goat polyclonal IL-22BP biotinylated antibody (R&D Systems, BAF1087) was added to each well in assay buffer at 0.5 μg/ml dilution and incubated for 1 h at 37°C followed by six washings.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: GRP94 ATPase activity is important for IL-22BPi1 and IL-22BPi2 secretion. GRP94 co-localizes with IL-22BPi1 and IL-22BPi2 but not IL-22BPi3. (Left) Confocal microscopy of HEK293 cells co-transfected with IL-22BP isoforms with the indicated GFP-GRP94 fusion constructs (depicted lower right); IL-22BP was stained using an anti-IL-22BP antibody (red, IL-22BP Ab 4), IL-2EX4 was stained with mouse anti-FLAG Ab, and the nucleus was stained using DAPI (blue). Secreted IL-22BP was measured by ELISA in the conditioned media (CM) of HEK293 cells transfected with individual expression vectors for IL-22BP isoforms together with the indicated GRP94 vectors (upper right) (mean ± SEM; n = 4; ** p < 0.01 by unpaired t -test).

Article Snippet: Antigen-purified goat polyclonal IL-22BP biotinylated antibody (R&D Systems, BAF1087) was added to each well in assay buffer at 0.5 μg/ml dilution and incubated for 1 h at 37°C followed by six washings.

Techniques: Activity Assay, Confocal Microscopy, Transfection, Construct, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BPi1 and IL-2EX4 induce unfolded protein response (UPR) genes. HEK293 cells were transiently transfected with IL-22BPi1, IL-22BPi2, IL-22BPi3, IL-2, IL-2EX4 or empty vector (EV) as control. Cells were collected at the indicated hours after transfection. (A) Expression of different genes related to ER function or UPR were analyzed by RT-qPCR. Each gene expression value is represented as fold change relative to the same time-point expression value of the EV condition and relative to the housekeeping gene GAPDH . Mean ± SEM of three independent experiments. All primers are listed in Supplementary Table . (B) GRP78 and GRP94 protein levels correlate with mRNA levels observed in (A) . Cell lysates (CL) were immunoblotted for FLAG, GRP94, KDEL and tubulin as loading control. (C) IL-22BPi1 and IL-2EX4 cause XPB1 splicing. XBP1 splicing was detected with conventional PCR for the indicated conditions and times. Un-spliced and spliced XBP1 are indicated as XBP1-u or XBP-1s respectively. (D) IL-22BPi2 secretion was not increased when co-expressed with different ratios of IL-22BPi1. HEK293 cells were co-expressed with different ratios of EV:IL-22BPi1:IL-22BPi2 expression plasmids. 48 h later, secreted IL-22BP in conditioned media (CM) was quantified by ELISA (mean ± SEM; n = 3). (E) Cell viability measured with alamarBlue was not compromised by any of the conditions in two different cell lines. Reduction of alamarBlue was measured after 48 h of transfection and assayed for the indicated times and cell lines. Values are represented as percentage of reduction in each condition relative to EV (mean ± SEM; n = 3).

Article Snippet: Antigen-purified goat polyclonal IL-22BP biotinylated antibody (R&D Systems, BAF1087) was added to each well in assay buffer at 0.5 μg/ml dilution and incubated for 1 h at 37°C followed by six washings.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Silencing of IL22RA2 in immature moDCs reduces GRP78 expression. (A) CD14 + monocytes were stained with DAPI (blue), trans-GOLGI (red) and IL-22BP (green, IL-22BP Ab 4). (B) Overall (best coverage, BC) and isoform-specific IL22RA2 expression levels in classical, intermediate and non-classical monocyte subpopulations (top) from four healthy donors (average ± SEM). Percentages of IL-22BP + monocytes found in each subpopulation by immunofluorescence microscopy (bottom) (mean ± SEM; n = 4; * p < 0.05, Kruskal-Wallis test for comparison of differences in IL22RA2 mRNA levels or IL-22BP + counts in the three subpopulations). (C) IL22RA2 expression levels in classical and intermediate immature moDCs at day 6 of cultivation. moDCs derived from non-classical monocytes showed high levels of cell death and had not experienced any changes in the shape of the cells. High Ct values of HK gene HPRT1 in qPCR compromised correct interpretation of IL22RA2 levels in this subgroup, which was therefore excluded from the graph. (D) Fold change relative to non-targeting siRNA control of IL22RA2 expression in CD14 + monocyte.-derived immature DCs on day 5 of cultivation following IL22RA2 silencing with Viromer Green at two concentrations of siRNA. (E) Silencing as described in (D) was not variant specific and both IL22RA2v1 and v2 were silenced to similar extent. (F) Scheme comparing efficiencies of four distinct silencing strategies on suppression of IL22RA2 mRNA (qPCR) and intracellular IL-22BP (ELISA) in immature CD14 + moDCs using 100 nM IL22RA2 siRNA or non-targeting siRNA in combination with Viromer Green. S, silencing, H, harvest. Bar diagrams represent fold change relative to non-targeting siRNA. (G) Effect of IL22RA2 silencing on GRP78 and ERp44 mRNA levels in CD14 + monocyte-derived immature DCs from healthy donors using silencing strategy B in (F) . CD14 + monocytes were cultivated in DM medium in the absence or presence of AM580 starting on day 0 up to the day of harvest. Average of fold change (±SEM) relative to non-silencing control over 6 (control) and 5 (AM580) independent measurements relative to the housekeeping gene HPRT1. * p < 0.03 by Wilcoxon test.

Article Snippet: Antigen-purified goat polyclonal IL-22BP biotinylated antibody (R&D Systems, BAF1087) was added to each well in assay buffer at 0.5 μg/ml dilution and incubated for 1 h at 37°C followed by six washings.

Techniques: Expressing, Staining, Immunofluorescence, Microscopy, Comparison, Derivative Assay, Variant Assay, Enzyme-linked Immunosorbent Assay