Journal: The Journal of Experimental Medicine
Article Title: ERBIN deficiency links STAT3 and TGF-β pathway defects with atopy in humans
Figure Lengend Snippet: ERBIN expression is reduced in STAT3 mut patient cells and in newly identified individuals with an ERBB2IP mutation. (A, top) Representative immunoblot showing ERBIN expression in primary dermal fibroblasts from control, STAT3 mut , and ERBB2IP mut patients. Lines indicate rearranged segments of one blot. (Bottom) Integrated densitometry of combined data comparing control ( n = 4) with STAT3 mut (SH2 mutants, n = 2; DNA-binding mutants, n = 2) or ERBB2IP mut ( n = 2) individuals. (B) ERBIN expression in naive (CD45RO − ) or memory (CD45RO + ) CD4 T cells from control ( n = 15), STAT3 mut ( n = 17), and ERBB2IP mut ( n = 3) patients. (C) Pedigree, chromatogram, and schematic of mutation (c.1588G > T p.[D530Y]) in ERBB2IP identified in a family with a dominant congenital allergic disorder with connective tissue features. LRR, leucine rich repeat domain; PDZ, PSD95-Dlg1-zo-1–like domain; SSID, SMAD–SARA interacting domain. (D) Representative immunoblot of GFP after MYC immunoprecipitation (IP) in 293T cells in which MYC-tagged wild-type STAT3 (MYC- STAT3 WT ) and GFP-tagged ERBIN-mutant (GFP- ERBB2IP mut ) or wild-type (GFP- ERBB2IP WT ) constructs were overexpressed in the presence or absence of STAT3 activation by IL-6. (E) Effect of wild-type ERBIN ( ERBB2IP WT ) or mutant ERBIN ( ERBB2IP mut ) on TGF-β–induced SMAD-reporter activity. AU, arbitrary units. Data are representative or combined from at least three independent experiments and represented as the mean ± SEM. Paired and unpaired two-tailed Student’s t tests and Mann-Whitney tests were used where appropriate. *, P
Article Snippet: For immunoprecipitation, after the indicated transfection and/or stimulation, 293T cells were lysed in immunoprecipitation lysis buffer (1× Mini protease inhibitor with EDTA [Roche], 20 mM Tris-HCl, pH 7.75, 140 mM NaCl, 1% Triton X-100, and 1 mM PMSF protease inhibitor cocktail [Thermo Fisher Scientific]), precleared, and immunoprecipitated using Protein A/G Plus agarose (Thermo Fisher Scientific) and monoclonal antibodies for SMAD2/3 or by using precoated sepharose beads for STAT3 and MYC (Cell Signaling Technology).
Techniques: Expressing, Mutagenesis, Binding Assay, Immunoprecipitation, Construct, Activation Assay, Activity Assay, Two Tailed Test, MANN-WHITNEY