Figure 6. IL-17B–IL-17RB signaling... | Rockefeller University Press

$Figure 6. IL-17B–IL-17RB signaling modulates CCL20, CXCL1, TFF1, and IL-8 expression through transcription factors NF-κB, ATF2, AML1, and AP1 via the TRAF6–ACT1–TAK1–ERK1/2 pathway. (A–D) Phospho-kinase array detected ERK1/2 phosphorylation in shLacZ-transduced or IL-17RB–depleted CFPAC-1 cells (A), and lenti-neo or IL-17RB overexpressing HPAC cells (B). Relative phosphorylation level of ERK1/2 protein to reference (Ref) is indicated (C and D). (E) Immunoblot analysis of ERK1/2 phosphorylation in shLacZ-transduced or IL-17RB–depleted CFPAC-1 and BxPC3 cells, and lenti-neo or IL-17RB overexpressing HPAC and SU.86.86 cells. GAPDH was used as a loading control. Relative phosphorylation (RP) level of ERK1/2 protein in IL-17RB–perturbed cells relative to control is indicated. (F) Immunoblot analysis of ERK1/2 and IKK phosphorylation in CFPAC-1 cells treated with 50 ng/ml rIL17B after pretreatment of 10 µM MEK/ERK inhibitor U0126 and/or 10 µM NF-κB inhibitor BAY117082 in a serum-free condition. GAPDH was used as a loading control. (G) mRNA expressions of CCL20, CXCL1, TFF1 and IL-8 were measured by qRT-PCR in CFPAC-1 cells treated rIL17B after pretreatment of 10 µM U0126 and/or 10 µM BAY117082 in a serum free condition. β-actin was used as an internal control. Relative expression of chemokines in rIL17B and/or kinase inhibitor treated cells relative to nontreated cells is indicated. (H) IB analysis of NF-κB subunit p65 in nuclear and cytosolic fractions of CFPAC1 cells. p84 was used as a loading control for nuclear fraction and GAPDH was used as a loading control for cytosolic fraction. (I) Reporter assay was performed using rIL17B-treated CFPAC-1 cells transfected with the promoter construct of CCL20, CXCL1, IL-8, or TFF1. Relative fold-change in luciferase activity (RLU) was shown. Data shown are means ± SD. *, P < 0.05; **, P < 0.01 (Student’s t test). (J) Diagram shows the predicted binding sites of NF-κB, ATF2, AML1 and/or AP1 on the −1-kb promoter regions of CCL20, CXCL1, TFF1, and IL-8. (K) Reporter assay using rIL17B-treated CFPAC-1 cells transfected with the segmented promoter construct of TFF1. Diagram showed the predicted binding sites of NF-κB, ATF2, and AML1 on the +1–250-bp promoter region of TFF1. (L) Time course assay using IB analysis to detect ATF2, AML1a, and c-Jun phosphorylation in CFPAC-1 cells treated with 50 ng/ml rIL17B. GAPDH was used as a loading control. Relative phosphorylation (RP) level of each protein at different time point relative to 0 time point is indicated. (M) Time-course ChIP of NF-κB, AP1, ATF2, or AML1 on the chemokine promoters in CFPAC-1 cells treated with 50 ng/ml rIL17B. Normal immunoglobulin G (IgG) was used as controls for promoter association. (N) Co-IP of IL-17RB, TRAF6, ACT1, and TAK1 in lenti-neo control, IL-17RB, or TRAF6-overexpressing HPAC cells. Normal IgG was used as a control. Relative enrichment (RE) level of IL-17RB-interacting proteins in rIL17B-treated cells at different time points relative to 0 min is indicated. Data shown are means ± SD. All experimental data were verified in at least two independent experiments.$

Figure 6.

IL-17B–IL-17RB signaling modulates CCL20, CXCL1, TFF1, and IL-8 expression through transcription factors NF-κB, ATF2, AML1, and AP1 via the TRAF6–ACT1–TAK1–ERK1/2 pathway. (A–D) Phospho-kinase array detected ERK1/2 phosphorylation in shLacZ-transduced or IL-17RB–depleted CFPAC-1 cells (A), and lenti-neo or IL-17RB overexpressing HPAC cells (B). Relative phosphorylation level of ERK1/2 protein to reference (Ref) is indicated (C and D). (E) Immunoblot analysis of ERK1/2 phosphorylation in shLacZ-transduced or IL-17RB–depleted CFPAC-1 and BxPC3 cells, and lenti-neo or IL-17RB overexpressing HPAC and SU.86.86 cells. GAPDH was used as a loading control. Relative phosphorylation (RP) level of ERK1/2 protein in IL-17RB–perturbed cells relative to control is indicated. (F) Immunoblot analysis of ERK1/2 and IKK phosphorylation in CFPAC-1 cells treated with 50 ng/ml rIL17B after pretreatment of 10 µM MEK/ERK inhibitor U0126 and/or 10 µM NF-κB inhibitor BAY117082 in a serum-free condition. GAPDH was used as a loading control. (G) mRNA expressions of CCL20, CXCL1, TFF1 and IL-8 were measured by qRT-PCR in CFPAC-1 cells treated rIL17B after pretreatment of 10 µM U0126 and/or 10 µM BAY117082 in a serum free condition. β-actin was used as an internal control. Relative expression of chemokines in rIL17B and/or kinase inhibitor treated cells relative to nontreated cells is indicated. (H) IB analysis of NF-κB subunit p65 in nuclear and cytosolic fractions of CFPAC1 cells. p84 was used as a loading control for nuclear fraction and GAPDH was used as a loading control for cytosolic fraction. (I) Reporter assay was performed using rIL17B-treated CFPAC-1 cells transfected with the promoter construct of CCL20, CXCL1, IL-8, or TFF1. Relative fold-change in luciferase activity (RLU) was shown. Data shown are means ± SD. *, P < 0.05; **, P < 0.01 (Student’s t test). (J) Diagram shows the predicted binding sites of NF-κB, ATF2, AML1 and/or AP1 on the −1-kb promoter regions of CCL20, CXCL1, TFF1, and IL-8. (K) Reporter assay using rIL17B-treated CFPAC-1 cells transfected with the segmented promoter construct of TFF1. Diagram showed the predicted binding sites of NF-κB, ATF2, and AML1 on the +1–250-bp promoter region of TFF1. (L) Time course assay using IB analysis to detect ATF2, AML1a, and c-Jun phosphorylation in CFPAC-1 cells treated with 50 ng/ml rIL17B. GAPDH was used as a loading control. Relative phosphorylation (RP) level of each protein at different time point relative to 0 time point is indicated. (M) Time-course ChIP of NF-κB, AP1, ATF2, or AML1 on the chemokine promoters in CFPAC-1 cells treated with 50 ng/ml rIL17B. Normal immunoglobulin G (IgG) was used as controls for promoter association. (N) Co-IP of IL-17RB, TRAF6, ACT1, and TAK1 in lenti-neo control, IL-17RB, or TRAF6-overexpressing HPAC cells. Normal IgG was used as a control. Relative enrichment (RE) level of IL-17RB-interacting proteins in rIL17B-treated cells at different time points relative to 0 min is indicated. Data shown are means ± SD. All experimental data were verified in at least two independent experiments.

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