Patient fibroblasts show different OSMR glycosylation and impaired signaling downstream of OSM. (A) Endogenous OSMR expression in fibroblast lysates from P1 and three healthy controls (HCs) with or without PNGase treatment. (B) Quantification of OSMR band intensities relative to GAPDH from A. (C and D) OSMR production of dermal fibroblasts from P1 and HCs treated with 25 µg/ml protein synthesis inhibitor (cycloheximide, CHX; 1M) for 24 h, and (D) quantification of WT OSMR bands and variant OSMR band intensities relative to GAPDH from C. Experiments shown in A and C were repeated twice with dermal fibroblasts from the patient and three HCs each. (E and F) Dermal fibroblasts from P1 and three HCs were stimulated with OSM (5 ng/ml) for 30 min. Whole-cell lysates were harvested for WB for the visualization of pSTAT3, STAT3, pSTAT5, STAT5, pERK1/2, ERK1/2, and GAPDH and quantification of WB band intensities shown in F. (G–J) Serum-starved dermal fibroblasts from P1 and three HCs were stimulated with IL-6 (100 ng/ml), IL-31 (100 ng/ml), or IL-11 (100 ng/ml) for 30 min. Whole-cell lysates were harvested for WB (G and I) for the visualization of pSTAT3, STAT3, and GAPDH and band intensities quantitated (H and J). (K) Dermal fibroblasts from P1 and HCs were stimulated with OSM (10 ng/ml) for 2 or 6 h. Total RNA was harvested for expression analysis by RT-PCR after 2 h for IL-6 and after 6 h for IRF7, CXCL10, and ICAM1. Data are representative of three independent experiments performed in quadruplicates. Statistics were calculated using the unpaired t test and comparing pooled samples from controls to patient. UT, untreated. * = P < 0.05, ** = P < 0.01. Source data are available for this figure: SourceData F3.