Western Blot Analysis of FOXP1 and FOXP4

Primary human neutrophils and HEK293 cells were solubilized in Lysis Buffer (20 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 3,5 mM sodium dodecyl sulfate, 13 mM deoxycholic acid) and extracted proteins were separated on a 4% to 15% Tris/Glycine/sodium dodecyl sulfate gel (Bio Rad). For transfer and blotting, we used a standard protocol. FOXP1 (#ab16645, abcam, 1:500) and FOXP4 (#ab17726, abcam, 1:500) primary antibodies were purchased from Abcam. The antirabbit IgG HRP-linked secondary antibody was purchased from Cell Signaling Technology and used at recommended concentrations. Signals of protein bands were detected using Clarity ECL chemiluminescent substrates (Bio-Rad) and ChemiDoc Imaging System (Bio-Rad). We quantified changes in protein levels relative to control using Image Lab software after normalization to β-actin or GAPDH (Cell Signaling Technologies), as indicated. Western blot images are representative of three biological replicates. Full western blots for FOXP1 and FOXP4 are provided in Figure S5 and correspond to the full-length FoxP1 and FOXP4 protein as described in (50 (link), 70 (link)–73 (link)).