Pierce s nitrosylation western blot kit

The assay was performed using the Pierce™ S-Nitrosylation Western Blot-Kit (Thermo Scientific™) following the manufacturer’s protocol. In brief, cell lysates were treated with ascorbate in HENS buffer for specific labeling with iodoTMT reagent after MMT pretreatment (Thermo Scientific™, #23011). Protein labeling was confirmed by Western blot using TMT antibody (Thermo Scientific™, #90075), according to standard procedures.

The Pierce S-Nitrosylation Western Blot kit (Cat no. 90105, Thermo scientific, USA) was used as per manufacturer’s instructions to estimate protein nitrosyl content in samples. Briefly, 1 M MMTS (2 µl) was added to sample (100 μl) with concentration of 1 μg/μl protein. The mixture was incubated for 30 min at room temperature. Six volumes of pre-chilled acetone were then used for protein precipitation. The precipitate was again resuspended using HENS buffer (100 μl). For each 50 μl sample aliquot, 1 μl labeling reagent was utilized. Further, 2 μl Sodium ascorbate (1 M) was added to the sample followed by incubation of 2 hr at room temperature. Then 10 μl reducing Laemmli sample buffer (5×) was added and heated at 100 °C for 5 min. The samples were then immunoblotted using Anti-TMT antibody (1:1000 in 5% NFDM in 1X TBST) and secondary Anti-mouse IgG-HRP conjugated antibody (1:20,000 in 5% NFDM in 1X TBST). Immunoblots were analyzed using a previously described method³⁶ .

Protein S-nitrosylation was measured by selective reduction and labeling of S-nitrosylated cysteines of the immunoprecipitated proteins with the Pierce S-Nitrosylation Western Blot Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. As the cysteine biotinylation is reversible, the samples were prepared without reducing agents.

Chemicals were of reagent or higher grade from Sigma-Aldrich (St Louis, MO, USA) unless otherwise specified. Anti-caspase-3 monoclonal (sc-136219), anti-calpain-1 monoclonal (sc-271313), agarose-conjugated (AC) anti-caspase-3 (sc-136219) and AC anti-calpain-1 antibodies (sc-271313) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorogenic calpain-1 substrate (208748) and colorimetric caspase-3 substrate (235400) were from Calbiochem (Darmstadt, Germany); colorimetric caspase-8 substrate (260-045-M005) and colorimetric caspase-9 substrate (260-081-M005) were from Enzo Life Sciences (Farmingdale, NY, USA). The enhanced chemiluminescence detection system was from Millipore Corporation (Bedford, MA, USA) and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); In situ Cell Death Detection Kit, Fluorescein (11-684-795-910) was from Roche; Cyclic GMP Enzyme-linked Immunosorbent Assay Kit was purchased from Cayman Chemical (Ann Arbor, MI, USA); whereas Pierce S-Nitrosylation Western Blot Kit was from Thermo Scientific. 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) was from Calbiochem (Darmstadt, Germany) and 17β-(3-hydroxy-1-propylamino)-1,3,5(10)-estratrien-3-ol, Prolame, was synthesized and chemical purity established as previously reported (Fernández-G et al., 1985 (link)).

Cell lysates were prepared by using a RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with Protease Inhibitor Cocktail (PIC, Sigma-Aldrich), Phenyl-methane Sulfonyl Fluoride (PMSF, Sigma-Aldrich), and Phosphatase Inhibitor Cocktail (PhoIC A and B, Sigma-Aldrich). Equal amounts of protein of each sample were analyzed using SDS-PAGE, electrophoretic transfer, immunoblotting, and chemiluminescence detection. Either 10% or 12% SDS-PAGE was used to isolate proteins with different molecular weights. The proteins were transferred to a nitrocellulose filter membrane (PALL). Antibodies and dilutions were as follows: anti-VEGFD, 1:1000; anti-PECAM-1, 1:1000; anti-LYVE1, 1:1000; anti-FLK-1, 1:1000; anti-VEGFR3, 1:1000; anti-HA, 1:1000; anti-Flag, 1:500; anti-Myc, 1:1000; anti-GAPDH, 1:5000; anti-GSNOR, 1:2000; The Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, Nebraska) was used to detect the immunoreactive signals. Western blot bands were quantified by measuring integrated density using the Image-J software (NIH, USA). Co-immunoprecipitation was performed through the cell lysates, and the supernatants were incubated with antibody and protein A/G PLUS-Agarose (Santa Cruz) overnight at 4℃. The bound proteins were eluted in the loading buffer and subjected to the SDS-PAGE. S-nitrosylated proteins were detected by Pierce™ S-nitrosylation Western Blot Kit (Thermo).