AMFR is required for TSLP signaling. (A and B) The protein and mRNA expression of AMFR in pulmonary macrophages after AM adoptive transfer. (C–F) The knockdown efficiency of AMFR shRNA in THP-1 cells and PBMCs was detected using Western blot (C and E) and qRT-PCR analysis (D and F). (G and H) Immunoblot analysis of phosphorylated (p-) STAT5, JAK1, JAK2, total STAT5, JAK1, JAK2, CIS, SOCS1, SOCS2, SOCS3, and β-actin in whole-cell lysates of BMDMs stimulated with TSLP (10 ng/ml) at various time points. The quantification of the blots was performed using ImageJ and is shown in a heatmap. (I and J) The knockdown efficiency of Cish shRNAs in BMDMs was detected using Western blot (I) and qRT-PCR analysis (J). (K) Immunoblot analysis of phosphorylated and total STAT5 protein levels in BMDMs transfected with lentivirus-mediated Cish shRNA or Scr shRNA, and 48 h later, stimulated with TSLP (10 ng/ml) at various time points. (L) Immunoblot analysis of phosphorylated and total STAT5 in HEK293T cells transfected with plasmids encoding IL-7R, TSLPR, STAT5, Flag-AMFR, and Myc-CIS, and 48 h later, stimulated with TSLP (10 ng/ml) for 60 min. The quantification of the blots was performed using ImageJ. (M and N) The knockdown efficiency of CIS in vivo was detected using Western blot (M) and qRT-PCR analysis (N). Data shown are representative of three independent experiments (A, C, E, I, and M). Each symbol represents one mouse, and data are presented as mean ± SD from one representative of three independent experiments (n = 5 mice per group per experiment; B and N). Each symbol represents data from an independent experiment, and data are presented as mean ± SD from one representative of three independent experiments (n = 3; D, F, and J–L). *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (Student’s t test, B, D, F, J, and N; two-way ANOVA with Tukey’s post hoc analysis, K; and one-way ANOVA with Tukey’s post hoc analysis, L). Source data are available for this figure: SourceData FS3.