Prestained protein marker

Luminol, bovine LPO, 3,3′,5,5′-tetramethylbezidine (TMB) and TMB liquid system, tyrosine, KBr, NaCl, 5-thio-2-nitrobenzoic acid (TNB), and 4-aminobenzoic acid hydrazide (ABAH) were purchased from Sigma-Aldrich (St. Louis, MO). Native human MPO was from Elastin Products Company, Inc. (Owensville, MO). pcDNA3.1, the CyQuant lactate dehydrogenase (LDH) cytotoxicity assay kit (catalog number C20300), and prestained protein markers were from Invitrogen (Carlsbad, CA). CM-Sepharose fast flow and Sephacryl S-300 were purchased from Cytiva (Marlborough, MA). Endoglycosidase H (Endo H) and peptide-N-glycosidase F (PNGase F) were from New England Biolabs (Ipswich, MA). Centrifugal filter devices (30-kDa cutoff) were from Millipore Corporation (Billerica, MA, USA). Anti-MPO monoclonal antibody (2C7) was purchased from Novus Biologicals (Centennial, CO). Pierce ECL Western blotting substrate was from ThermoFisher Scientific (Grand Island, NY).

SDS-PAGE analysis was carried out as previously reported by Laemmli with some modifications [22 (link)]. A vertical slab gel of 1.5 mm thickness was used, and protein was separated by SDS-PAGE with 12% acrylamide separating gel and 4% stacking gel (Bio-Rad Laboratories, Mini-PROTEAN 3 Cell). SDS-PAGE patterns were analyzed using Image Lab software (Bio-Rad, 6.0.1, USA) to obtain the molecular weight and relative content of proteins (relative to M-9). Prestained protein markers (10-180 kDa, Thermo Fisher, Waltham, Ma, USA) were used as standard.

Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 g/L D-glucose, L-glutamine, 25 mM HEPES without phenol red, dimethylsulphoxide, Fura-2 AM, pluronic F-28, Triton X-100, CaCl_2, MnCl_2, and antagonist ICI-118,551, and 2-D quant kit were obtained from Sigma- Aldrich, USA. Chemicals used in the preparation of 2-D cell lysis buffer and sodium dodecyl sulfate-polyacrylamide were from HiMedia, India. NFA was procured from PureSynth Research Chemicals, Canada. The Femto-Plus Chemiluminescence detection kit and pre-stained protein markers were acquired from Thermo Fisher Scientific, USA. EDTA-free protease and broad-spectrum phosphatase inhibitor cocktail including Tyr, Ser/Thr phosphatases were obtained from Roche, Germany. The antibodies used in this study were as follows: rabbit anti-β-2-AR antibody purchased from BoosterBio, USA; mouse anti-α-tubulin antibody, mouse phosphotyrosine antibody, and PSA-FITC obtained from Sigma Aldrich, USA; HRP- and FITC-conjugated swine anti-rabbit and goat anti-mouse antibodies were from Dako-Agilent, USA.

Cells were lysed in a RIPA buffer containing 10 mM Tris HCl, pH 7.5, NaCl 150 mM, and Triton 1% supplemented with Protease Inhibitor Cocktails (Sigma Aldrich) and the protein content was measured by a colorimetric assay (BIO-RAD) as described in^{71 (link)}. Protein cell lysates (20 μg) were separated by SDS-PAGE under denaturing conditions, transferred onto a nitrocellulose filter (BIO-RAD), blocked with 5% milk and probed with primary antibodies: mouse anti-PARP1 (1:500), rabbit anti-procaspase-3 (1:500) and mouse anti-βactin (1:1000) (Santa Cruz Biotechnology). After primary antibody incubation, the blots were washed and incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Thermo Fisher Scientific). The proteins recognized by Abs were detected by ECL (Amersham biosciences) and exposed to Las4000 (GE Healthcare Life Sciences) and the relative band intensities were determined using ImageQuant software (GE Healthcare Life Sciences). Pre-stained protein markers (Thermo Fisher Scientific) were used as molecular size standards. All experiments were performed in triplicate.

Whole-cell protein extracts were separated on SDS-PAGE before electrophoretic transfer onto a nitrocellulose membrane (Amersham Biosciences). The membranes were incubated with primary antibodies diluted in a blocking buffer overnight and then with relevant secondary antibodies for one hour. Antibodies recognizing LRP6 (diluted 1:1000) was purchased from Abcam, tRFP (diluted 1:2000) from Evrogen, β-Actin antibody (diluted 1:2000) from Oncogene Research Products, horseradish peroxidase-conjugated anti-rabbit IgG (diluted 1:2000) from Vector Laboratories. Immunocomplexes were visualized by using ECL (Amersham). The molecular weight of proteins was estimated with pre-stained protein markers (ThermoScientific). Band intensities were determined by densitometric analysis of immunoblots with Molecular Imager FX and Quantity One software (BioRad).

Cells were lysed in a buffer containing 10 mM Tris HCl, pH 7.5, NaCl 150 mM, and Triton 1% supplemented with Protease Inhibitor Cocktails (Sigma Aldrich, Saint Louis, MO, USA) and the protein content was measured by a colourimetric assay (BIO-RAD, Hercules, CA, USA). Protein cell lysates (20 μg) were separated by SDS-PAGE under denaturing conditions, transferred onto a PVDF filter (BIO-RAD, Hercules, CA, USA), blocked with 5% milk and probed with primary antibodies: anti-ERK2, anti-phospho-ERK, anti-ICAM1, anti-PARP1, anti-pan-cathepsin and anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-AKT and anti-phospho-AKT (Ser-473) (Cell Signalling Technologies, Danvers, MA, USA). After primary antibody incubation, the blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (ThermoFisher Scientific; Waltham, MA, USA). The proteins recognized by Abs were detected by ECL (Amersham biosciences; Little Chalfont, UK). Pre-stained protein markers (ThermoFisher Scientific; Waltham, MA, USA) were used as molecular size standards. All experiments were performed in triplicate.