Sample buffer

RIPA buffer protein extracts were diluted 1:2 in 0.1× sample buffer (bio-techne; 042-195) and run on the Jess Simple Western system using a 12-230 kDa separation module (bio-techne) according to manufacturer’s instructions. Antibodies and dilutions used for capillary-based immunoblotting (Jess) are detailed in Supplementary Data 1d. Protein peak areas were quantified using Compass software (bio-techne) and normalized to internal protein loading controls within each capillary.

HeLa and N2a cells were harvested and lysed in Pierce RIPA buffer (ThermoFisher; #89901) with cOmplete proteinase inhibitor cocktail (Roche; #11697498001). Total protein levels were determined using a Bradford assay (ThermoFisher; #23236); Samples were diluted in 0.1× sample buffer (Bio-techne) and a total of 1.2 μg protein per sample was loaded for antibody staining. The primary antibody for human and mouse JAK1 was monoclonal anti-JAK1 (Cell Signaling; #50996) in a 1:500 dilution; and for human and mouse housekeeping β-actin was monoclonal anti-β-actin (Cell Signaling; #4970) in a 1:500 dilution. Secondary antibodies used were: Anti-mouse detection module (Bio-techne; #DM-002) and anti-rabbit detection module (Bio-techne; #DM-001). The assay was performed as described by the ProteinSimple protocol using the 16–230 kDa separation module (Bio-techne; SM-W004) on a Wes system (Bio-techne). For in vivo studies, footpad skin biopsies were mechanically homogenized in 500 μL Pierce RIPA buffer proteinase inhibitor cocktail, the processed skin homogenate was centrifuged at 10,000×g for 5 min, and the clear supernatant was collected for Wes ProteinSimple assay. The primary antibody for mouse JAK1 was monoclonal anti-JAK1 (Cell Signaling; #50996) in a 1:500 dilution; and for mouse housekeeping β-actin was monoclonal anti-β-actin (Cell Signaling; #4970) in a 1:250 dilution.