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

Figure 3.

Figure 3

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.