β actin

Cells were harvested with RIPA buffer and incubated on ice for 30 min. The concentration of total protein was quantified by the BCA Protein Assay kit (Applygen, Beijing, China). Equal protein lysates were loaded for each sample and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: p-STAT3 antibody1 : 1000 (CST, USA), STAT3 antibody 1 : 1000 (CST, USA), and β-actin 1 : 6000 (Applygen, China). The membranes were incubated with anti-rabbit IRDye 800CW secondary antibody 1 : 10000 (LI-COR Biosciences, USA), and the bands were scanned on an Odyssey Infrared Imaging System (LI-COR Bioscience).

Lung tissues were first ground into a single-cell suspension. Obtained lung cells and MH-S cells were lysed in RIPA buffer (Applygen, Beijing, China) for protein extraction. After determining protein concentrations with a bicinchoninic acid colorimetric assay kit (Applygen), protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membranes (MilliporeSigma, Burlington, MA, USA). Membranes were blocked in 1× Tris-buffered saline containing Tween with 5% w/v non-fat dry milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against p-p65 (1:1,000; CST), p65 (1:1,000; CST), p-p38 (1:1,000; CST), p38 (1:1,000; CST), p-ERK1/2 (1:1,000; CST), ERK1/2 (1:1,000; CST), p-JNK (1:1,000; CST), JNK (1:1,000; CST), p-Akt (1:1,000; CST), Atk (1:1,000; CST), α₁-AR (1:1,000; Abcam), and β-actin (1:5,000; Applygen). After three washes, membranes were incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized using a chemiluminescence image analysis system (Tanon Science and Technology, Shanghai, China).

BCA protein quantification kit (Applygen), sodium hydrosulfide (Sigma-aldrich), L-cysteine (Sinopharm), propargylglycine (Aladdin), Anti-eNOS Rabbit Antibody (Santa Cruz), Anti-p-Akt Mouse Antibody (Santa Cruz), Anti-t-Akt Mouse Antibody (Santa Cruz), Anti-cPKCβII Rabbit Antibody (Santa Cruz), HRP-labeled Goat Anti-Mouse IgG (Applygen), HRP-labeled Goat Anti-Rabbit IgG (Applygen), β-actin, Mouse mAb (Applygen), Multi-functional enzyme immunoassay (Thermo), Gel imager (Bio-Rad), Membrane and cytosol protein extraction kit (Beyotime).

Total protein was obtained from cells after lysed in RIPA buffer. The Western blot was performed as described in our previous work.32 The information of primary antibodies was HA (Cat No. 3724, Cell Signaling Technology), SFRP2 (Cat No. 06‐004, millipore), KDM2A (Cat No. ab31739, abcam), BCOR (Cat No. 12107‐1‐AP, proteintech), phosphorylated β‐catenin (Cat No.2009, Cell Signaling Technology), GAPDH (Cat No. C1312, Applygen) and β‐actin (Cat No. C1313, Applygen).

Total protein extraction and SDS-polyacrylamide gel electrophoresis procedures were performed as described in a previous study [22 (link), 23 (link)]. Immune complexes on membranes were incubated with horseradish peroxidase‐conjugated anti‐ rabbit or anti‐mouse IgG (Promega, Madison, WI) and visualized with SuperSignal reagents (Pierce, Rockford, IL). The primary antibodies used in this study were anti‐EREG (Cat No. 93815; Cell Signaling Technology, Boston, MA, USA), mouse monoclonal anti‐HA (Clone No. C29F4; Cat No. MMS‐101P; Covance, Princeton, NJ, USA), anti‐phospho‐p38 MAPK (Cat No. 4631; Cell Signaling Technology, Boston, MA, USA), anti‐p38 MAPK (Cat No. 8690; Cell Signaling Technology, Boston, MA, USA), anti‐phospho‐Erk1/2 (Cat No. 4377S; Cell Signaling Technology, Boston, MA, USA), and anti‐Erk1/2 (Cat No. 4695S; Cell Signaling Technology, Boston, MA, USA). The primary monoclonal antibody used as a housekeeping control was a monoclonal antibody against β‐actin (Cat No. C1313; Applygen, Beijing, China).

After the protein quantification with BCA kits, the protein levels of TIPE2,
MyD88, TRIF, TRAF3 and TRAF6 in splenic tissue from different groups were
analyzed by SDS-PAGE electrophoresis. The protein was then transferred to
polyvinylidene fluoride membrane after electrophoresis. The membranes were
blocked with 5% skim milk diluted in TBST, followed by an overnight incubation
with primary antibodies against β-actin (1:2000 dilution; Applygen), TIPE2
(1:1000 dilution; protein tech), MyD88 (1:1000 dilution; cell signaling
technology), TRIF (1:1000 dilution; Bioworld), TRAF3 (1:1000 dilution;
Bioworld), TRAF6 (1:1000 dilution; Bioworld). The membranes were subsequently
incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000
dilution; Applygen). Finally, images were examined with an ImageQuant LAS 4000
imager, and strip density was analyzed by Quantity One software.

Protein extraction and gel electrophoresis were performed as described in our previous work.^{57 (link)} The primary antibodies were as follows: WDR5 (Cat No. sc-393080, Santa Cruz, USA), MLL1 (Cat No. 14197 S, Cell Signaling Technology, USA), Flag (Cat No. 66008-4-Ig, Proteintech, USA), HES1 (Cat No. ab 108937, Abcam, UK), H3K4me3 (Cat No. 9751 s, Cell Signaling Technology, USA), Histone H3 (Cat No. SC-56616, Santa Cruz, USA) and β-actin (Cat No. C1313, Applygen, China).