Horseradish peroxidase conjugated anti mouse and anti rabbit igg

Stenodactylin was purified from the caudex of Adenia stenodactyla Harms as previously described (Stirpe et al., 2007 (link)); the purity grade was > 99%. CellTiter 96^® Aqueous Non-Radioactive Cell Proliferation Assay was obtained from Promega Corporation (Madison, WI, USA). PhosSTOP - Phosphatase Inhibitor and complete ULTRA protease inhibitor cocktail were purchased from Roche Applied Science (Penzberg, Germany). The primary antibodies against phospho-SAPK/JNK (Thr183/Tyr185; 81E11), p38, phospho-p38 (Thr180/Tyr182; 12F8), p44/42 MAPK (Erk1/2), COX IV, and the secondary horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The primary antibody against caspase 3 and p-ERK 1/2 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Antibodies were diluted following manufacturer’s instructions. Phospho flow cytometry was performed with Alexa Fluor^® 647 conjugate mouse antibodies against phospho-p38 (Thr180/Tyr182; clone 36/p38), phospho-JNK (Thr183/Tyr185; clone N9-66), phospho-ERK1/2 (Thr202/Tyr204; clone 20A), and IgG isotype κ control (clone MOPC-21) purchased from BD transduction Laboratories (Heidelberg, Germany) (Mercatelli, 2015 ).

Western blotting was performed as previously described^{17 (link)} using rabbit primary antibodies to SIRT3 (#5490, Cell Signaling Technology, Danvers, MA, USA), OPA1 (#80471, Cell Signaling Technology), cleaved caspase-3 (Asp175) (Cell Signaling Technology) and PINK1 (Sigma-Aldrich) diluted 1:1000. Mouse monoclonal primary antibodies to α-tubulin (DM1A) (Cell Signaling Technology) and β-actin (A5441, clone AC-15) (Sigma-Aldrich) were diluted, respectively, 1:1000 and 1:5000 before use. Horseradish peroxidase conjugated anti-mouse and anti-rabbit IgG (Cell Signaling Technology) secondary antibodies were used at 1:2,000 dilution. A semi quantitative measurement of band intensity was performed using the Carestream Molecular Imaging Software (Carestream Molecular Imaging, New Haven, CT, USA) and Fiji^{36 (link)}.

Chemicals were obtained from the following sources. BSA (fatty acid-free), the adenylyl cyclase inhibitor LRE1 and the Ca²⁺ ionophore A23187 were purchased from Sigma. Protein kinase inhibitor PKI 14–22 amide myristoylated (sPKI) was obtained from Tocris. All other chemicals were purchased from Cayman Chemicals (Ann Arbor, MI). Anti-Acetyl αTubulin and the antibodies against the deacetylases were obtained from Santa Cruz Biotechnology. Anti-phosphotyrosine (anti-pTyr) monoclonal antibody (clone 4G10) was obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit monoclonal anti-phospho-PKA substrates (anti-pPKA substrates) (clone 100G7E), anti-Acetyl Lysines (9441 S) antibodies and Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG, were purchased from Cell Signaling Technology (Danvers, MA). Alexa 546 anti-rabbit, Alexa 488 anti-mouse and Slow-Fade Light reagents were obtained from Molecular Probes (Eugene, OR). Anti-PKAc was purchased from BD Biosciences (clone 5B) and anti-PKARII from Abcam (#38949). Anti-mouse and anti-rabbit IgG light chain (211-032-171 and 115-035-174 respectively) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Cells were lysed in RIPA buffer (R&D Systems) containing protease inhibitors (Sigma-Aldrich) and total protein was determined by BCA (Thermo Fisher Scientific). Lysates or supernatants were separated by SDS-PAGE (NuPAGE, Thermo Fisher Scientific) and blotted onto nitrocellulose, nytrane membranes (GE healthcare, Chicago, IL, USA). Anti-mouse caspase-1, full-length and activated p20 fragment (mAb Casper-1, Adipogen Life Sciences, Liestal, Switzerland), ASC (anti-Asc, pAb (AL177), Adipogen Life Sciences), NLRP3 (mAB Cryo-2, Adipogen Life Sciences), IL-1 ß (anti-mIL-1β R&D Systems) were used as primary and horseradish-peroxidase-conjugated anti-rabbit and anti-mouse IgG (both Cell Signaling Technology, Beverly, MA, USA) as secondary antibodies. Chemiluminescent substrate (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) was used for visualization.

HPMCs were harvested from culture dishes, and proteins were extracted using radioimmunoprecipitation assay buffer containing Halt protease inhibitor (Pierce). Immunoblotting was performed with primary antibodies against fibronectin, ST2 and β‐galactosidase (all from Santa Cruz Biotechnology); E‐cadherin, Snail, collagen 4, alpha‐smooth muscle actin (αSMA), p65, phosphorylated (p‐)p65, CCN1, GAPDH and IL‐33 (all from Abcam); and β‐actin (Sigma‐Aldrich). Equal amounts (30 μg) of extracted protein were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to an Immobilon‐FL 0.4‐μm polyvinylidene difluoride membrane (Millipore). Horseradish peroxidase‐conjugated anti‐rabbit and antimouse IgG (both from Cell Signaling Technology) were used as secondary antibodies. Labelled proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech; ECLTM PRN 2106) using a Gel Doc 1000 imager with Multi‐Analyst v.1.1 software (Bio‐Rad).