S nitrosylated protein detection assay kit

The S-Nitrosylated Protein Detection Assay Kit (Cayman Chemicals, Ann Arbor, USA) was used and involved the following steps: (1) RBC lysis and blocking of free thiol (SH) groups; (2) cleavage of S-NO bonds present in the sample; and (3) biotinylation of SH groups. Protein concentration of the samples was determined using the DC-Protein Assay Kit (BioRad, Munich, Germany) and 50 µg protein was loaded into the lanes of a 3–8% Tris-acetate gel (BioRad, Munich, Germany). Proteins were separated for 1 h with constant 90 mA according to their charge and mass in a 1 x XT Tricine running buffer (BioRad, Munich, Germany). Separated proteins were transferred to a polyvinylidene difluoride membrane (0.45-mm pore size) and the membrane was blocked in 2% BSA in TBS containing 0.1% Tween^® 20 prior to incubation with horseradish peroxidase (dilution 1:2000). The immunoreactive bands were developed using an enhanced chemiluminescence kit (Thermo Scientific, Darmstadt, Germany) and band intensities were calculated in ImageJ software (National Institutes of Health, Bethesda, USA) (adapted to [24 (link)]).

We used the S-nitrosylated Protein Detection Assay Kit (Cayman Chemical, Cat: 10006518) to detect nitrosylated H-Ras in the immunoprecipitated H-Ras preparation. Biotin-labeled NO-H- Ras was analyzed by the Western blot assay [immunolabeled by enhanced chemiluminescence (ECL) Streptavidin HPR, Amersham Biosciences, UK)] followed by visualization with ECL (Amersham Biosciences, UK) and analysis by densitometric scanning. Total H-Ras was detected after stripping the nitrocellulose membrane and incubation with the anti-H-Ras primary antibody (Abcam, dilution: 1:4000). Immunolabeled bands were visualized using ECL and analyzed by densitometric scanning.

Expression of S-nitrosylated proteins in cells was detected by the biotin switch method using an S-Nitrosylated Protein Detection Assay Kit (CAYMAN CHEMICAL COMPANY) in combination with fluorophore labeling and visualization by confocal microscopy [9 (link)].

S-nitrosylated protein was detected using a biotin switch assay according to the instructions of a S-nitrosylated Protein Detection Assay Kit (Cayman Chemical, Ann Arbor, MI, USA)^{17 (link)}. Firstly, the free sulfhydryl group in the sample was blocked with methyl methanethiol sulfonate (MMTS). Secondly, the excess blocking agent was removed by acetone precipitation, and the thiol nitroso group (-SNO) was selectively reduced to free thiol by ascorbate. Finally, biotin-HPDP was added to label the newly synthesized thiol group. The biotin-labeled protein was adsorbed by streptavidin-agarose, subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE).

Protein was extracted according to the manufacturer's specification S-nitrosylated Protein Detection Assay Kit (Cayman, USA), which is based on the “Biotin-switch” method as described previously [12 (link)].

As described previously (Zhou et al, 2019 (link)), proteins were extracted according to the manufacturer’s specification S-Nitrosylated Protein Detection Assay Kit (Cayman, USA), which is based on the “Biotin-switch” method.

Proteins were extracted according to the manufacturer's specification S‐Nitrosylated Protein Detection Assay Kit (Cayman, USA) which is based on the ‘Biotin‐switch’ method as described previously.¹¹ Akt activity was determined in a kinase reaction using recombinant GSK‐3α as substrate.²⁴ Two µl GSK‐3α protein/ATP mixture was added into 50 µl kinase buffer and incubated at 30°C for 14 hours. Phosphorylation of the GSK‐3α can be analysed by Western blot analysis using the phospho‐GSK‐3α‐specific antibody. The level of phospho‐GSK‐3α was calculated to represent Akt activity.