Anti stat1

Total protein was isolated from the lung tissue cleared of blood using RIPA buffer (Beyotime) containing protease (Beyotime) and phosphatase (Roche, Basel, Switzerland) inhibitors. The Pierce BCA Protein Assay Kit (Thermo Scientific, USA) was used to estimate the protein concentration. Lysates were adjusted to a concentration of 3 μg/μL. The protein samples were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes (Millipore, Germany). The membranes were blocked with 5% skim milk for 1 h at room temperature in TBST buffer and probed with the following primary antibodies: anti-STAT1, anti-phospho-STAT1, anti-STAT3, anti-phospho-STAT3 (Affinity, 1 : 500), and anti-β-actin (CST, 1 : 1000) overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (CST, 1 : 2000). Protein bands were visualized using enhanced chemiluminescence (ECL). β-Actin was used as a loading control. ImageJ software was used to analyze the optical density of the protein bands.

Proteins were separated using 12% SDS-PAGE and were then transferred to a polyvinylidene fluoride (PVDF) membrane and probed with their respective antibodies. The T47D and SK-BR-3 cells were homogenized in RIPA lysis buffer (Meilunbio, Shanghai, China) containing protease inhibitor mixture on ice for 30 min, and centrifuged for 10 min at 12,000 rpm in a 4 ˚C Eppendorf microfuge (Thermo, USA). Loading buffer was added to the protein lysate and boiled for 5 min. The following primary antibodies were used: Anti-VWCE (1:1000; Affinity Biosciences), anti-STAT1 (1:50; Affinity Biosciences), and anti-MS4A4A (1:100; Affinity Biosciences) were used as primary antibodies. MXB was used for secondary antibody detection. The immunocomplexes were then visualized by chemiluminescent imaging.

Total proteins were extracted from spleen tissues with a extraction Kit (KeyGen BioTECH, Nanjing, China), and the protein samples (30 μg) was separated onto a 8% SDS-PAGE gel (STAT1), 10% SDS-PAGE gel (JAK1, GATA1, EPOR), and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 2 h, and then washed with Tris buffered saline Tween (TBST) for three times, 5 minutes each. Thereafter, PVDF membranes were incubated with primary antibodies anti-JAK1(1:1000), (Abcam, Cambridge, USA), anti-STAT1(1:500), anti-GATA1(1:500), anti-EPOR (1:500), and anti-GAPDH (1:1000), (Affinity, California, USA), at 4°C overnight. All membranes were washed with TBST buffer for 3 times, 10 minutes each, and then incubated with secondary antibodies at room temperature for 2 h, respectively. All immunoreactive bands were visualized with a ChemiDoc MP Imaging System (Bio-rad, USA) and analyzed by Gel-Pro32 software.