Stat3

Total protein from cells or tissues was extracted and quantified. Twenty‐five micrograms of proteins was separated by 10% polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane. Skim milk (5%) was used to block non‐specific antigen binding, and then primary antibodies were added (RASSF1A, ab23950, Abcam; YAP, #14074, Cell Signaling Technology; pS127‐YAP, #13008; Cell Signaling Technology; Cyclin A, sc‐596, Santa Cruz Biotechnology; Cyclin B1, 55004‐1‐AP, Proteintech; Cyclin D1, sc‐246, Santa Cruz Biotechnology; Cyclin E, sc‐25303, Santa Cruz Biotechnology; CDK1, WL02373, Wanleibio; cleaved‐caspase‐3, WL02348, Wanleibio; Bcl‐2, WL01556, Wanleibio; BAX, 50599‐2‐lg, Proteintech; p73, WL01604, Wanleibio; p53, sc‐126, Santa Cruz Biotechnology; p21, WL0362, Wanleibio; AKT, WL0003b, Wanleibio; p‐AKT, WLP001a, Wanleibio; ERK, WL01864, Wanleibio; p‐ERK, WLP1512, Wanleibio; STAT3, WL03207, Wanleibio; p‐STAT3, WLP2412, Wanleibio; NF‐κB P65, WL01980, Wanleibio; LATS1/2, YT2543, ImmunoWay Biotechnology; p‐LATS1/2, YP1222, ImmunoWay Biotechnology; MST1/2, 37462, Signalway Antibody; p‐MST1/2, bs‐3294R, Bioss; β‐actin, sc‐47778, Santa Cruz Biotechnology; Histone H3, Wanleibio) and incubated at 4°C overnight. Next, secondary antibodies were added, followed by incubation at room temperature for 1 hour and subsequent exposure to an X‐ray film.

After treatment with LPS for a particular period, peritoneal macrophages from MED1^fl/fl or MED1^ΔMac mice were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Then, the lysates were centrifuged, and the supernatant was harvested. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Protein samples (40 μg) were subjected to 8%–15% SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore), and immunoblotted with antibodies against p65 (1 : 1000, Cat. No. 8284, Cell Signaling Technology, Beverly, MA, USA); p-p65 (1 : 1000, Cat. No. WL02169, Wanleibio, Shenyang, China); STAT1 (1 : 1000, Cat. No. 9172, Cell Signaling Technology); p-STAT1 (1 : 1000, Cat. No. WL02276, Wanleibio); STAT3 (Cat. No. 4904, Cell Signaling Technology); and p-STAT3 (1 : 1000; Cat. No. 9145; Cell Signaling Technology). β-Actin (1 : 5000, Cat. No. AP0060, Bioworld Technology Inc., Louis Park, MN, USA) was used as a loading control.

Immunoblotting analysis was performed as described previously (Kamat et al., 2013 (link)). Briefly, approximate 100 mg frozen lung tissue powder (n = 5 in each group) was lysed in 1 mL RIPA buffer containing 100 mM PMSF to extract pulmonary total proteins. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Equal amount of proteins (10 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane (BioRad). The blots were blocked with 5% (w/v) skimmed milk in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween- 20, pH 7.5) for 2 h. Then blots were incubated with primary antibodies overnight at 4°C. Subsequently blots were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 37°C. After ECL substrates were added, the blots were analyzed using light imaging system (Tanon 5200, China). Before each step, the blots were washed five times for 5 min with TBST.
In this study, β-actin (Proteintech) was determined as an internal control of the western blot. The primary antibodies were activator of transcription 1 (STAT1) (Beyotime Biotech, China), STAT3 (Wanleibio, China), p-STAT1 Y701 (Cell Signaling Technology) and p-STAT3 Y705 (Cell Signaling Technology). All secondary antibodies were peroxidase-conjugated affinipure goat anti-rabbit IgG (Proteintech).

PHrodo™ Green E. coli BioParticles™ was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Stattic, Fludarabinum, C29, TAK-242 were bought from MCE (Monmouth Junction, NJ, United States). BAY 11-7085, SP610025, PD184352, LY294002, AZD0530 and Y-27632 were purchased from Selleckchem (Houston, TX, United States). Mouse IL-1β, IL-6, TNF-α Quantikine ELISA Kit, Cytochalasin D were bought from R&D Systems (Minneapolis, MN, United States). Mouse myeloperoxidase/MPO ELISA Kit was purchased from MultiScience (Lianke) Biotech Co., Ltd. (Hangzhou, China). p-STAT3 (Tyr705), p-STAT1 (Tyr701), p-p65(Ser536), p-Src (Tyr416), p-Lyn (Tyr507), p-SAPK/JNK (Thr183/Tyr185), p-p38 (Thr180/Tyr182), p-ERK1/2 (Thr202/Tyr204) were bought from Cell Signaling Technology (Beverly, MA, United States). STAT3, STAT1, p65, Src, Lyn, JNK, p38, ERK1/2 were purchased from wanleibio (Shenyang, China). β-actin was purchased from Bioworld Technology (Bloomington, MN, United States). FITC anti-mouse F4/80 antibody (clone BM8), APC anti-mouse CD64 (FcγRI) antibody (clone X54-5/7.1), PE anti-mouse CD16/32 antibody (clone 93), purified anti-mouse CD16/32 antibody (clone 93) and Ultra-LEAF™ Purified anti-mouse CD64 (FcγRI) antibody (Clone W18349F) were purchased from Biolegend (San Diego, CA, United States). Rhodamine phalloidin and DAPI were purchased from Beyotime (Nanjing, China).

Total protein was collected by Radio-immunoprecipitation assay buffer with phosphatase inhibitor, and quantified by a bicinchoninic acid protein assay kit (Solarbio). Then, the protein was separated by 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Wanleibio), transferred to 0.45-µm polyvinylidene fluoride membranes (Millipore), and blocked with 5 % skim milk (BD) for 2 h at room temperature. The membranes were incubated overnight with the appropriate primary antibodies (YAP1:1/1000, Cell Signaling Technology (CST)#14,074; P-YAP1 (Ser127): 1/1000, CST#13,008; HK2: 1/1000, CST#2867; PKM2: 1/1000, CST#4053; STAT3: 1/1000, Wanleibio#WL01836; P-STAT3(Ser727): 1/1000, Wanleibio#WLP2412; VEGFA:1/1000, Proteintech#66,828; β-Actin: 1/10,000, Proteintech#60008-1-Ig) at 4 °C. The next day, the membranes were washed with Tris-buffered saline containing Tween 20 (TBST), then incubated with the appropriate secondary antibodies (1/10,000, CST) for 1 h at room temperature. Subsequently, the membranes were washed with TBST and the bands were detected using an ECL Western Blot Detection Kit (Wanleibio).