Figure 1. SOCS2 protein is ubiquitinated and degraded during pulmonary inflammation.

(A) Immunoblot analysis of leukocyte pellets from ARDS patients or control serum samples. Data represent mean ± SEM, (n = 7–10; ***P < 0.001 compared with control, Mann-Whitney U test). (B) Cell type clustering results of control lung tissue cells from single-cell RNA sequencing of SOCS2 transcript. (C) Immunoblotting following CHX (50 μg/mL), MG132 (20 μM), or leupeptin (50 μg/mL) treatment for the indicated times with ectopic expression of V5-tagged SOCS2 in MLE cells. (D) Immunoblotting following overexpression of ubiquitin (Ubi) in MLE cells. (E) UbiCRest analysis of SOCS2 ubiquitination following immunoprecipitation from MLE cells; digestion supernatant is shown below, with deubiquitinase enzymes indicated by asterisks. Ctrl, control; CD, catalytic domain. (F) Immunoblot analysis following expression of SOCS2-V5 and ubiquitin lysine-to-arginine mutants. (G) Immunoblotting analysis of MLE cells following SOCS2-HIS and HA-ubiquitin expression. Following HIS PD, eluate was immunoblotted for HA ubiquitin signal. (H) qPCR analysis of MLE cells treated with LPS for the indicated times. Data represent mean values ± SEM (n = 3; NS, P > 0.05 compared with 0 hours; Student’s 2-tailed unpaired t test). (C–G) Data are representative of n = 2–3 independent experiments.