Skip to main content
. 2021 Oct 9;297(5):101295. doi: 10.1016/j.jbc.2021.101295

Figure 1.

Figure 1

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.