Eecl western blot kit

S-nitrosylated Protein Detection Assay Kit (Cayman, USA) based on the “Biotin-switch” method [49 (link)] was used for S-nitrosothiol detection according to the manufacture's instruction. All steps were done with minimal light exposure. Briefly, 100–250 μg protein lysates were extracted from SKOV3 cells treated with DETA-NONOate (50 μM) or L-NAME (1 mM). PKM2 protein was purified by immunoprecipitation (TR0064.0, ThermoFisher Scientific, Waltham, MA) and its free thiols were blocked by blocking agent. S-nitrosothiols in the protein were reduced to free thiol(s) and subsequently covalently labeled with maleimide-biotin. After concentration measurement and dilution with SDS loading buffer, the labeled proteins were denatured at 95°C. Samples were sent to immunoblotting with HRP-detected reagent and visualized by the eECL Western Blot Kit (#P90720, Millipore).

After adding phenylmethanesulfonyl fluoride (PMSF) to inhibit protease activity and radio immunoprecipitation assay (RIPA) lysate buffers, rat hippocampus tissue was crushed by an ultrasonic cell disintegrator (Scientz). Then, the rat hippocampus was centrifuged at 10464.5 g for 10 min, the supernatant was extracted, and the protein concentration was determined by a BCA protein assay kit. Before the sample is taken, the protein mixture is boiled for 10 min in a 100°C water bath to denaturate it. 10% SDS polyacrylamide separation gel was prepared, and then, gel electrophoresis (120 V, 90 min) (Bio‐Rad) was carried out. After electrophoresis, it was transferred to polyvinylidene fluoride (PVDF) membrane (200 mA, 75 min). Then, the membrane was immersed in 5% skimmed milk powder or BSA and blocked for 1 hr. After closure, the primary antibody was added, including P‐tau^S396 (1:10,000), total tau (1:5,000), SYN (1:20,000), PSD95 (1:1,000), Bax (1:2,500), Bcl‐2 (1:2,500), P‐CREB (1:5,000), CREB (1:1,000), and beta‐actin (1:50,000), and incubated overnight at 4 C. Next day, the goat anti‐rabbit IgG H&L (HRP) (1:5,000) was incubated at room temperature for 2 hr; then, the chemiluminescent liquid was prepared with eEcl Western Blot kit (Millipore), and the exposure imaging was performed in the chemiluminescent imager (ProteinSimple).

S-nitrosylated Protein Detection Assay Kit (Cayman, USA) based on the “Biotin-switch” method was used for S-nitrosothiol detection according to the manufacturer’s instruction. All steps were done with minimal light exposure. Briefly, 100–250 μg protein lysates were extracted from cells or the digested tumor tissues. Its free thiols were blocked by blocking agent. S-nitrosothiols in the protein were reduced to free thiol(s) and subsequently covalently labeled with maleimide-biotin. Then we purified all S-nitrosylated proteins using Streptavidin Agarose Resins. After concentration measurement and dilution with SDS loading buffer, the labeled proteins were denatured at 95 °C. The membrane was blocked with 5% bovine serum albumin and probed with the appropriate primary antibodies: rabbit anti-PFKM (55028-1-AP, 1: 500 dilutions). Samples were sent to immunoblotting with HRP detected reagent and visualized by the eECL Western Blot Kit (#P90720, Millipore). Western blot detection of the content of the SNO-PFKM which was enriched by streptavidin.