Image lab software

For western blot, the tissues were lysed in RIPA buffer, which containing 50 mmol/L Tris/HCl (pH 8.0), 150 mmol/L NaCl, 1% Nonidet-P40, 1% sodium deoxycholate, 0.1% SDS, 0.1 mmol/L DTT, 0.05 mmol/L PMSF, 0.002 mg/ml aprotinin, 0.002 mg/ml leupeptin, and 1 mmol/L NaVO3. The BCA protein assay was used to determine the protein concentration of each supernatant. Equal amounts of protein were loaded and separated by 12% SDS-PAGE, and then, the proteins were transferred onto polyvinylidene difluoride membranes. The membranes were incubated overnight with appropriate primary antibodies at 4 °C, which including CD3G, GNAS, MAP3K8, NGFR, PRKG2, PTPN22, JAK 1, JAK 2, JAK 3, p-Stat 1, p-Stat 3, p-Stat 5, p-Stat 6, IKKα, IKKβ, p-IKKα/β, NF-κB, p-NF-κB, IκBα, p-IκBα, CD3ε (CD3-12), p-Lck, p-PLCγ1 (Tyr783), p-SLP-76, p-Src, p-Zap-70, PI3K(p85), Akt, p-Akt, mTOR, p-mTOR. Furthermore, the membranes were further incubated with HRP-conjugated secondary antibodies (anti-rabbit or mouse immunoglobulin G) for 1 h at 25 °C. The immunoreactivity was detected by enhanced chemiluminescence (ECL) (Bio-Rad, USA), and β-actin was conducted as a loading control. The Image Lab™ software was performed for quantitative analysis, and all reagents were purchased from Cell Signaling Technology of USA.

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)).