Ab32520

Whole-cell lysates were obtained from mouse mφs at the specified time points by lysing the cells in complete radioimmunoprecipitation lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 2 mM sodium orthovanadate, and 1× protease inhibitor mixture [Fisher, BPE9706-1] in PBS). Phosphatase inhibitor was added to the lysis buffer when harvesting cell lysates for pStat6 detection. Cell lysate (10 μg) was subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The blot was incubated with either goat polyclonal anti-Pu.1 (1:200, D-19, sc-5949; Santa Cruz Biotechnology), rabbit monoclonal anti-Stat6 (1:2000, YE361, ab32520; Abcam), or rabbit polyclonal anti-pStat6 (1:1000, Tyr⁶⁴¹, ab195647; Abcam). Protein was detected using ECL (Thermo Fisher Scientific).

Peritoneal macrophages were washed with ice‐cold PBS and homogenized on ice using RIPA lysis buffer (Beyotime) supplemented with complete protease inhibitor (Beyotime). Samples were loaded on 10% SDS‐PAGE and transferred onto PVDF membranes (Bio‐Rad, Shanghai, China). Membranes were blocked with 5% non‐fat dry milk and incubated with primary antibodies against iNOS (ab129372; Abcam, Cambridge, USA), Arg‐I (ab91279; Abcam), p‐STAT6 (ab54461; Abcam), STAT6 (ab32520; Abcam), respectively. β‐actin was used as control. The intensities of the corresponding protein bands were evaluated by densitometry via Image J (National Institutes of Health, USA) Protein expression was assessed relative to β‐actin or indicated protein.

The expression levels of STAT1, STAT4, STAT6 and the corresponding phosphorylated proteins in lung digests were analyzed by Western blot analysis. Cell lysates (40 μg) were separated by 10% sodium dodecyl sulfate-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, USA). After incubation in a blocking buffer containing 5% skim milk in TBST (12.5 mM Tris–HCl pH 7.5, 68.5 mM NaCl, and 0.1% Tween 20) for 1 h, the blots were incubated overnight with primary antibodies including rabbit anti-STAT1 (D1K9Y, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT1 (Tyr701, Cell Signaling Technology, USA), rabbit anti-STAT4 (C46B10, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT4 (ab28815, Abcam, UK), rabbit anti-STAT6 (ab32520, Abcam, UK), and rabbit anti-phosphorylated STAT6 (ab28829, Abcam, UK). An anti-mouse GAPDH antibody was used as a loading control. The blots were washed with TBST, incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit Ig (Jackson ImmunoResearch) and then developed with an enhanced chemiluminescence (ECL) substrate solution (Millipore).

STAT6 overexpression vector or NC vector was transfected in M0 macrophages for 24 h. Then, cells were under M2 polarization. Forty-eight hours later, cells were harvested for chromatin immunoprecipitation (ChIP) assays. ChIP assays were performed following the methods described previously [52 (link)] using antibodies against STAT6 (ab32520, Abcam), a positive control antibody (RNA polymerase II), and a negative control non-immune IgG. The immunoprecipitated DNA was cleaned, released, eluted, and used for real-time PCR. The fold-enrichment (FE) was calculated as previously described [52 (link)].

Samples were homogenized in lysis buffer (pH 7.4) containing a protease inhibitor cocktail (HY-K0010, MedChenExpress), phosphatase inhibitor cocktail III (HY-K0023, MedChenExpress), 50 mM Tris, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% NP-40 (Sigma-Aldrich). The protein concentration was determined using a BCA Protein Assay kit (Tiangen Biotech). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and the samples were blotted onto a polyvinylidene fluoride (PVDF) membrane. Antibodies against DPP4 (ab28340, Abcam), F4/80 (28463-1-AP, Proteintech), CD206 (18704-1-AP, Proteintech), p44/42 MAPK (Erk1/2) (137F5) rabbit mAb (4695, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, Cell Signaling Technology), signal transducer and activator of transcription 6 (STAT6) (ab32520, Abcam), and Phospho-STAT6 (phospho Y641) (ab263947, Abcam) were used at a dilution of 1:2000. Secondary antibody binding and detection were performed according to standard protocols using an enhanced chemiluminescence (ECL) detection reagent (Bio-Rad).

Rabbit monoclonal anti–Tyr⁶⁴¹–phosphorylated STAT6 (p-STAT6) antibody (ab54461) and rabbit monoclonal anti–STAT6 antibody (ab32520) were bought from Abcam (Cambridge, MA). Rabbit monoclonal anti–Tyr⁷⁰¹–phosphorylated STAT1 (p-STAT1) antibody (#9167) and rabbit polyclonal anti–STAT1 antibody (#9172) were bought from Cell Signaling Technology (Beverly, MA). Goat polyclonal anti–platelet–derived growth factor (PDGF) receptor α antibody (AF1062) was bought from Roche (Nutley, NJ). Rat monoclonal anti-Mac3 antibody (550292) was bought from BD Pharmingen (San Diego, CA). Bleomycin was bought from Nippon Kayaku (Tokyo, Japan). 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine- 2-carboxylic acid methylamide (BLZ945) was purchased from Chemshuttle (Jiangsu, China). Rat monoclonal IgG_2a anti-mouse IL-13 antibody (38213) and control rat monoclonal IgG_2a (54447) were bought from R&D systems (Minneapolis, MN). 4’6-diamidino-2-phenylindole (DAPI) (D1306) was purchased from Molecular Probes (Eugene, OR). FastStart Universal SYBR Green Master was bought from Roche (Nutley, NJ). All other reagents were bought from Wako (Osaka, Japan) unless otherwise specified. An S1PR2–specific antagonist (S1PR2i) [18 ] was provided by Ono Pharmaceutical Co.