Ruxolitinib and fedratinib inhibit TNF-α-mediated STAT1 and STAT3 with attenuation of c-Jun and NF-κB. A. Representative western blots of nuclear lysates from human umbilical vein endothelial cells (HUVEC) treated with 0.4 μM ruxolitinib or 1 μM fedratinib JDB for 30 minutes followed by addition of 10 ng/mL TNF-α for 4 hours. Nuclear extracts collected and separated on 10% Tris-Hcl gel, transferred to PVDF membrane, and followed by total protein stain and immunoprobes against phospho-antibodies. Membranes were stripped and re-probed for unphosphorylated forms. B. Densitometry values from 5 different westerns blots probed for phospho-STAT1 normalized to total STAT1. C. Densitometry values from 5 different western blots for phospho-STAT3 normalized to total STAT3. D. Densitometry values for 5 different westerns blots probed for phospho-C-JUN normalized to total protein values. E. Densitometry values for 5 different western blots probed for phospho-p65 NF-κB to total protein. Error bars represent mean ±SE of means. p values from analysis of variance testing with appropriate post-hoc multiple comparison testing. ns, not significant. * p < .05, ** p < .01, *** p < .001.