Journal: Journal of clinical immunology
Article Title: IL4Rα and IL17A Blockade Rescue Autoinflammation in SOCS1 Haploinsufficiency
doi: 10.1007/s10875-023-01635-z
Figure Lengend Snippet: SOCS1 T68fs fails to inhibit cytokine signaling by loss-of-expression. A SOCS1 mRNA levels by qPCR in A549 cells transduced with either empty vector (EV), WT SOCS1, or T68fs SOCS1. P-values for individual comparisons from statistical testing shown above. B Western blotting for Flag-tagged SOCS1 transfected into HEK239T cells. Immunoblotting by polyclonal SOCS1 antibodies or anti-Flag antibody. For anti-SOCS1 immunoblotting, the band present in all conditions below ~23kDa is non-specific. The horizontal arrow indicates the predicted molecular weight of T68fs SOCS1. The asterisk indicates the non-specific band. C Western blot for STAT1/2 phosphorylation by IFN-I stimulation of SOCS1-transfected HEK239T cells. D Western blot for STAT1 phosphorylation by IFN-II stimulation of SOCS1-transduced A549 cells. E Western blot for STAT6 phosphorylation by IL4 in SOCS1-transduced A549 cells. F ISRE-inducible Luciferase activity in SOCS1-transfected HEK239T cells stimulated with IFN-I. G Interferon-stimulated gene induction in SOCS1-transfected HEK239T cells stimulated with IFN-I.
Article Snippet: Antibodies against the following proteins were used: SOCS1 (ThermoFisher), Flag M2 (Sigma Aldrich), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phosphor-Tyr 689 STAT2 (Cell Signaling Technology), phospho-Tyr 641 STAT6 (Cell Signaling Technology), USP18 (Cell Signaling Technology), and β-actin (Cell Signaling Technology).
Techniques: Expressing, Transduction, Plasmid Preparation, Western Blot, Transfection, Molecular Weight, Phospho-proteomics, Luciferase, Activity Assay