Anti perk

Total ovary lysates were separated on SDS-PAGE. After electrophoresis, the protein was transferred to PVDF membrane. The membranes were blocked with 5% skimmed milk and incubated with anti-GRP78 (1:1000, #11587–1-AP, Proteintech, Hubei, China), anti-PARP1 (1:4000, #66520-1-lg, Proteintech), anti-PAR (1:1000, #4335-MC-100, Bio-techne, Minneapolis, USA), anti-mTOR (1:1000, #20657-1-AP, Proteintech), anti-p-mTOR (1:800, #sc-293133, Santa cruz, Dallas, Texas, USA), anti-KitL (1:1000, #bs-0545R, Biorbyt, Hubei, China), anti-p-IRE1α (1:800#, bs-16698R, Biorbyt), anti-IRE1α (1:1000, #27528-1-AP, Proteintech), anti-ATF4 (1:1000, #10835-1-AP, Proteintech), anti-p-PERK (1:800, #Orb336657, Biorbyt), anti-PERK (1:1000, #24390-1-AP, Proteintech), anti-HA-tag (1:1000, #30701ES60, YEASEN), anti-FLAG-tag (1:1000, #30501ES60, YEASEN) and anti-GAPDH (1:4000, #ab8245, Abcam, Cambridge, UK) antibodies overnight at 4 °C. After TBST washing, the membranes were treated with the corresponding peroxidase-conjugated secondary antibody, and then the signal was detected with an Enhanced Chemiluminescence Detection Kit (#32106, Thermo Scientific, Waltham, MA, USA) on Tanon 4500 gel imaging system (Shanghai, China).

The upper lobe of left lung without lavage was immediately fixed with 10% formaldehyde for 48 h, embedded in paraffin, sliced into 4 μm thick sections, and stained with hematoxylin and eosin solution (H&E) or Alcian blue periodic acid Schiff (AB/PAS) for histopathologic examination and goblet cell metaplasia of bronchial epithelium.
For analyzing the effects of GFDHP on mucus secretion, the Mucin5ac (Muc5ac) protein expression levels in the airway epithelium were performed by immunohistochemistry (IHC) analysis [18 (link)]. Change in the MAPK pathway in the airway was investigated on the basis of detecting Muc5ac regulatory proteins including extracellular signal-regulated kinase (ERK)/p-ERK/specificity protein1(SP1) through IHC. After antigen was repaired and endogenous peroxidase was inactivated and blocked, the sections were incubated overnight at 4°C with anti-Muc5ac (Abcam, USA, dilution 1 : 200), anti-ERK (dilution 1 : 200), anti-p-ERK (dilution 1 : 500), and anti-SP1 (dilution 1 : 200) antibodies (Proteintech, China) successively. The sections were then incubated with horseradish peroxidase- (HRP-) conjugated second antibodies and developed with 3, 3-diaminobenzidine tetrahydrochloride. The degree of brown stains reflected the expression levels of the target proteins.

Total ovary lysates were separated on SDS-PAGE. After electrophoresis, the protein was transferred to PVDF membrane. The membranes were blocked with 5% skimmed milk and incubated with anti-GRP78 (1:1000, #11587–1-AP, Proteintech, Hubei, China), anti-PARP1 (1:4000, #66520-1-lg, Proteintech), anti-PAR (1:1000, #4335-MC-100, Bio-techne, Minneapolis, USA), anti-mTOR (1:1000, #20657-1-AP, Proteintech), anti-p-mTOR (1:800, #sc-293133, Santa cruz, Dallas, Texas, USA), anti-KitL (1:1000, #bs-0545R, Biorbyt, Hubei, China), anti-p-IRE1α (1:800#, bs-16698R, Biorbyt), anti-IRE1α (1:1000, #27528-1-AP, Proteintech), anti-ATF4 (1:1000, #10835-1-AP, Proteintech), anti-p-PERK (1:800, #Orb336657, Biorbyt), anti-PERK (1:1000, #24390-1-AP, Proteintech), anti-HA-tag (1:1000, #30701ES60, YEASEN), anti-FLAG-tag (1:1000, #30501ES60, YEASEN) and anti-GAPDH (1:4000, #ab8245, Abcam, Cambridge, UK) antibodies overnight at 4 °C. After TBST washing, the membranes were treated with the corresponding peroxidase-conjugated secondary antibody, and then the signal was detected with an Enhanced Chemiluminescence Detection Kit (#32106, Thermo Scientific, Waltham, MA, USA) on Tanon 4500 gel imaging system (Shanghai, China).

METH was provided by Ningbo Public Security Bureau. N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich. All drugs were dissolved in saline (0.9% NaCl) and administered to cells directly or via intraperitoneal injection. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DMSO were purchased from MedChemExpress. The BrdU cell proliferation kit was purchased from BioVision. The primary and secondary antibodies for Western blotting, including anti-Ras, anti-β-actin, anti-ERK, anti-pERK, anti-MEK, and anti-pMEK, were purchased from Proteintech. Polyvinylidene fluoride, blocking buffer, and ECL were purchased from Millipore. The Active Ras Pull-Down and Detection Kit was purchased from Thermo Fisher Scientific.

Tissues were homogenized in ice-cold lysis buffer (50 mM Tris HCl, pH 7.5, 0.5% Nonidet P-40, 150 mM NaCl, 2 mM EGTA, 1 mM Na₃VO₄, 100 mM NaF, 10 mM Na₄P₂O₇, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin)^{11 (link)}. Tissue extracts were then immunoblotted with the following primary antibodies: anti-p-eIF2α, anti-EIF2α, anti-GCN2, anti-p-HSL, anti-HSL, anti-IRE1α, and anti-p-PKA substrates (1:1000, Cell Signaling Technology, MA, USA); anti-UCP1 and anti-ATF6 (1:1000, Abcam, Cambridge, UK); anti-TH (1:1000, Merck Millipore, Frankfurter, GER); anti-ATF4 and anti-TRB3 (1:500, Santa Cruz Biotechnology, CA, USA), anti-p-GCN2 (1:500, Biorbyt, Cambridge, UK); anti-CHOP, anti-ATF4, anti-PERK, and anti-XBP1s (1:1000, Proteintech, Hubei, P.R.C.); anti-p-IRE1α (1:1000, Epitomics, CA, USA); anti-p-PERK (1:1000, Signalway Antibody, MD, USA); anti-BIP and anti-β-actin (1:2000, Sigma, MO, USA).