Data for : Voltage-gated potassium channel proteins and stereoselective S-nitroso-L-cysteine signaling
Method 1: L-CSNO binding to proteins after native-PAGE separation The mouse brain membrane fraction (above) was resuspended in HEPES buffer. A BCA (Pierce) protein assay was performed and 50 µg protein was run on two native-PAGE gels (TGX, Bio Rad). One gel was incubated for 30 min in the dark with 50 µM L-CSNO in S-nitrosothiol buffer (10 mM Tris-HCl, pH 6.0, 150 mM NaCl). The second gel was incubated for 30 min in the dark with 50 µM L-CSNO and 100 µM each S-phenyl-cysteine (L-CSφ) and S-methyl-cysteine (L-CSMe) in S-nitrosothiol buffer. Each gel was then rinsed with water and incubated in the dark with 40 µM diaminofluoroscein (DAF) 2 (Cayman Chemicals) for 10 min in the dark (RT) (24). Gels were then imaged on Chemidoc (Bio Rad; Hercules, CA) using the fluorescein setting and bands cut out for mass spectrometry proteomics (see below) that were seen without, but not with, the S-methyl and S-phenyl substituted cysteine co-incubations (21). Method 2: L-CSNO affinity chromatography L-CSNO affinity columns were prepared as follows. L-Cysteine was coupled to AminoLink Plus (Pierce/ThermoFisher; Waltham, MA) resin according to the manufacturer’s protocol. In control experiments, aminoLink Plus was used without reaction with L-cysteine. Briefly, L-cysteine was dissolved in coupling buffer A (0.1 M sodium citrate, 0.05 M sodium carbonate, pH 10) at a concentration of 100 mM and incubated with 0.4 ml, previously washed, AminoLink resin in a small (0.5 ml) column for 4 hours at room temperature. The resin was then washed with coupling buffer B (0.1 M phosphate. 0.15 M sodium chloride, pH 7.2) and incubated for 4 hours at room temperature with 100 mM cyanoborohydride in coupling buffer B. Active sites were then blocked with quenching buffer (1 M Tris-HCl, pH 7.4). Resin was then washed with 1 M NaCl and then incubated for 10 min with 30% ethyl nitrite (EtONO) in ethanol at room temperature in the dark (25). It was then washed with 100% ethanol followed by 1M NaCl. Excised mouse brain homogenate was briefly centrifuged and the supernatants were then incubated with prepared Aminolink columns for 30 min at room temperature. The columns were then washed with wash buffer and bound protein eluted with Laemmli buffer and elution buffer (0.1 M Glycine, pH 3.5). Eluate was concentrated and used for SDS-PAGE gel (TGX, BioRad; Hercules, CA) followed by Coomassie staining. Bands found in samples from + L- Cysteine columns but not in – L-cysteine columns were excised and analyzed further by MS proteomics. Biotin substitution analysis for NO-substituted cysteine’s in Kv proteins CHO cells expressing all three Kv proteins were plated on in 6 wells of a six well plate. Once confluent, cells were incubated with no treatment, 500 µM L-CSNO, or 500 µM L-CSNO plus 10mM leucine for two minutes. Wells were then washed with cold PBS and lysed in HEN buffer (250mM HEPES pH7.7, 1mM EDTA, 0.1mM Neocuproine). After biotin switch, samples went to LC-MS.