Journal: The Journal of Biological Chemistry

Article Title: Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

doi: 10.1016/j.jbc.2021.101295

Figure Lengend Snippet: The N-terminal region of murine p40 from Y31 to T38 mediates binding to murine IL-12Rβ1. A , alignment of murine and human p40 N-terminal amino acids with analyzed mp40 deletion variants. B , crystal structure of the IL-23:IL-23R:IL-12Rβ1 complex (PDB 6WDQ ). Insets show zoomed in views on the area surrounding residues D36-Y38. D36-Y38 are depicted in orange stick representation. p40 and IL-12Rβ1 residues in contact with D36-Y38 are displayed in stick representation. p40 mutations D36K, W37K and Y38K were visualized in the second inset . C , Western blot analysis of secreted murine HIL-23 variants from transfected CHO-K1 cells. D , cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of four are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗ p ≤ 0.001. E , analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of two. F , co-IP of FLAG-tagged murine HIL-23 variants (wild-type, ΔM23-D29, ΔM23-P39) and full-length mIL-12Rβ1. The position of mIL-12Rβ1 and HIL-23 variants is indicated by arrows . One of two independent experiments is shown. G , co-IP of FLAG-tagged murine HIL-23 variants (wild-type, ΔM23-D29, ΔM23-P39) and full-length mIL-23R. The position of mIL-23R and HIL-23 variants is indicated by arrows . One of two independent experiments is shown.

Article Snippet: Biotinylated mIL-23R (BAF1686), hIL-23R (BAF1400), mIL-12Rβ1 (BAF1998), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), mp35/mp40 (BAF419), mp19 (BAF1619), hp40 (BAF219) mAbs, and streptavidin-HRP (DY998) were from R&D Systems.

Techniques: Binding Assay, Western Blot, Transfection, Cell Culture, Activation Assay, Co-Immunoprecipitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

doi: 10.1016/j.jbc.2021.101295

Figure Lengend Snippet: W37 of murine p40 is important for binding of HIL-23 to murine IL-12Rβ1. A , alignment of murine and human p40 N-terminal amino acids Y31 to P39. Single, double, and triple substitutions within mp40 are highlighted in red . B , Western blot analysis of secreted murine HIL-23 variants from transfected CHO-K1 cells. C , cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of three are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗ p ≤ 0.001, ns not significant. D , analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of three. E , co-IP of FLAG-tagged murine HIL-23 variants (wild-type, D36K, W37K and T38K) and full-length mIL-12Rβ1. The position of mIL-12Rβ1 and HIL-23 variants is indicated by arrows . One of two independent experiments is shown. F , co-IP of FLAG-tagged murine HIL-23 variants (wild-type, D36K/W37K/T38K, D36K/W37K, W37K/T38K) and full-length mIL-12Rβ1. The position of mIL-12Rβ1 and HIL-23 variants is indicated by arrows . One of two independent experiments is shown.

Article Snippet: Biotinylated mIL-23R (BAF1686), hIL-23R (BAF1400), mIL-12Rβ1 (BAF1998), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), mp35/mp40 (BAF419), mp19 (BAF1619), hp40 (BAF219) mAbs, and streptavidin-HRP (DY998) were from R&D Systems.

Techniques: Binding Assay, Western Blot, Transfection, Cell Culture, Activation Assay, Co-Immunoprecipitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

doi: 10.1016/j.jbc.2021.101295

Figure Lengend Snippet: Alanine substitutions of p40 D36, W37 and T38 impaired HIL-23 activity. A , alignment of murine and human p40 N-terminal amino acids Y31 to P39. Single and triple substitutions within mp40 are highlighted in red . B , Western blot analysis of secreted murine HIL-23 variants from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. C , analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of three. D , cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of the indicated cytokines (0.04–20% conditioned cell culture supernatant of transfected CHO-K1 cells). The results of one representative experiment of four are shown. Error bars represent S.D. for technical replicates.

Article Snippet: Biotinylated mIL-23R (BAF1686), hIL-23R (BAF1400), mIL-12Rβ1 (BAF1998), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), mp35/mp40 (BAF419), mp19 (BAF1619), hp40 (BAF219) mAbs, and streptavidin-HRP (DY998) were from R&D Systems.

Techniques: Activity Assay, Western Blot, Transfection, Activation Assay, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

doi: 10.1016/j.jbc.2021.101295

Figure Lengend Snippet: Murine p40 with D36K/W37K/T38K substitutions interacts with murine p19 but not with murine IL-12Rβ1. A , structure of hIL-23 extracted from the structure of the IL-23:IL-23R complex structure (PDB 5mzv ). A flexible linker sequence connecting p40 and p19 is indicated. B , Western blot analysis of secreted murine HIL-23, mp19 and mp40 variants from transfected CHO-K1 cells. The position of mp19 and mp40 variants is indicated by arrows . C , cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of three are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗ p ≤ 0.001. D , analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of two. E , co-IP of FLAG-tagged murine mp19 coexpressed with either mp40 or mp40D36K/W37K/T38K and full-length mIL-12Rβ1 or mIL-23R. The position of mIL-23R and mIL-12Rβ1 is indicated by arrows . One of two independent experiments is shown.

Article Snippet: Biotinylated mIL-23R (BAF1686), hIL-23R (BAF1400), mIL-12Rβ1 (BAF1998), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), mp35/mp40 (BAF419), mp19 (BAF1619), hp40 (BAF219) mAbs, and streptavidin-HRP (DY998) were from R&D Systems.

Techniques: Sequencing, Western Blot, Transfection, Cell Culture, Activation Assay, Co-Immunoprecipitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

doi: 10.1016/j.jbc.2021.101295

Figure Lengend Snippet: Substitution of the amino acid sequence D36/W37/T38 diminished antagonistic properties of homodimeric p40. A , Western blot analysis of secreted mp40 and mp40D36K/W37K/T38K conditions. In total, 20 μl conditioned supernatant of transiently transfected CHO-K1 was separated by SDS-PAGE under (non-)reducing conditions, and proteins were visualized by Western blotting with a mp40-specific antibody. B , co-IP of FLAG-tagged murine p40 variants (wild-type, D36K/W37K/T38K) and full-length mIL-23R or mIL-12Rβ1. The position of mIL-23R and mIL-12Rβ1 is indicated by arrows . One of two independent experiments is shown. C , co-IP of FLAG-tagged human p40 variants (wild-type, D36K/W37K/Y38K) and full-length hIL-12Rβ1. One of two independent experiments is shown. D , cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 7.5 ng/ml murine HIL-23 with or without 2 μg/ml mp40. The results of one representative experiment of two are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗ p ≤ 0.001, ns not significant. E , cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells. The cells were cultured for 3 days in the presence of 5 ng/ml murine HIL-12 with or without 2 μg/ml mp40. The results of one representative experiment of three are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗ p ≤ 0.001, ns not significant. F , cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 7.5 ng/ml murine HIL-23 with or without 2 μg/ml mp40D36K/W37K/T38K. The results of one representative experiment of two are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗ p ≤ 0.001, ns not significant. G , cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells. The cells were cultured for 3 days in the presence of 5 ng/ml murine HIL-12 with or without 2 μg/ml mp40D36K/W37K/T38K. The results of one representative experiment of three are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, ns not significant. H and I , analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R or Ba/F3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells were washed and starved for at least 4 h. One hour prior to stimulation 2 μg/ml mp40 or mp40D36K/W37K/T38K have been added. Cells were stimulated with the indicated cytokines (7.5 ng/ml HIL-23, 5 ng/ml HIL-12) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of two.

Article Snippet: Biotinylated mIL-23R (BAF1686), hIL-23R (BAF1400), mIL-12Rβ1 (BAF1998), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), mp35/mp40 (BAF419), mp19 (BAF1619), hp40 (BAF219) mAbs, and streptavidin-HRP (DY998) were from R&D Systems.

Techniques: Sequencing, Western Blot, Transfection, SDS Page, Co-Immunoprecipitation Assay, Cell Culture, Activation Assay