Protease inhibitors and phosphatase inhibitors

Following treatment of cells with the indicated reagent(s), protein samples were extracted using ice-cold lysis buffer (Sigma-Aldrich) containing protease inhibitors and phosphatase inhibitors (Sigma-Aldrich). An aliquot containing 15 µg of extracted protein was analyzed by western blotting for expression level of proteins of interest with the corresponding specific antibodies (Supplementary Table S1), as previously described^{48 (link)}.

Lung tissues were homogenized in RIPA buffer containing protease inhibitors and phosphatase inhibitors (Sigma Aldrich) and centrifuged at 12,000× g for 10 min. Supernatants were collected and total protein levels were measured using BCA protein assay (Thermo Scientific). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA), with protein expression levels being analyzed using antibodies against phospho-extracellular-signal-regulated kinase (ERK)1/2 (CST#4370, Cell Signaling, Farmingdale, NY, USA), ERK1/2 (CST#4695, Cell Signaling), phospho-JUN N-terminal kinase (JNK) 1/2 (CST#9255, Cell Signaling), JNK 1/2 (CST#9258, Cell Signaling), phospho-p38 MAPK (CST#4511, Cell Signaling), p38 MAPK (CST#8690, Cell Signaling), phospho-STAT3 (CST#9145, Cell Signaling), STAT3 (CST#9139, Cell Signaling), and β-actin (GTX629630, GeneTex, Irvine, CA, USA). Blots were washed three times with TPBS containing 0.05% Tween-20 and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) (Santa Cruz, Santa Cruz, CA, USA) for 1 h at room temperature. Signals were visualized with enhanced chemiluminescence (Thermo Scientific) and imaged using a Bio-Rad ChemiDoc XRS⁺ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) after an additional wash.

Freshly isolated RPTCs were suspended in protein lysis buffer (1% Triton X-100, 150 mM NaCl, and 10 mM Tris-HCl, pH 7.4; 1 mM EDTA; 1 mM EGTA; 2 mM sodium orthovanadate; 0.2 mM phenylmethylsulfonyl fluoride; 1 mM HEPES, pH 7.6) containing protease inhibitors and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Following sonication, protein was quantified using a bicinchoninic acid assay, subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with primary and secondary antibodies. Membranes were detected using chemiluminescence and processed using ImageJ (NIH, Bethesda, MD) software.

For S-100 cytosolic extraction, 30×10⁶ cells (primary lymphocytes, Raji or Jurkat) were incubated with ice-cold S-100 buffer (20mM HEPES-KOH at pH 7.5, 10mM KCL, 1.5mM MgCl₂, 1mM EDTA, 1mM EGTA, 1mM DTT supplemented with protease inhibitors and phosphatase inhibitors (Sigma, St Louis, MO). Cells were disrupted using a Dounce homogenizer and the pellet was centrifuged at 1000g for 10 minutes at 4⁰C. Supernatants were further centrifuged at 100,000g for 1 hr, and the resulting supernatant (S-100 cytosol) was stored at -80^oC or used immediately. The S-100 pellet was dissolved in 1X RIPA lysis buffer with protease and phosphatase inhibitors and stored at -80⁰C or used immediately. For cell free caspase-3 activation, S-100 cytosol (120μg) from cells treated with MPO (5μM), MPO-Zn (200μM), LY30 (25μM), NaN₃ (5μg/ml), etoposide (5μΜ) or daundorubicin (0.5μl/μl) were incubated in the presence or absence of 1mM dATP and 4μM cytochrome C (Sigma, St Louis, MO) for 30min at 37⁰C. Caspase-3 activity was then measured by using fluorogenic substrate DEVD-AFC as described previously [40 (link)].