Lambda protein phosphatase lambda pp

After overnight dialysis to remove phosphatase inhibitors, protein samples (40μl) were incubated with 5μL of 10X NEBuffer for Protein MetalloPhosphatases (PMP), 5μL of 10mM MnCl₂ and 1μl of Lambda Protein Phosphatase (Lambda PP, New England Biolabs) at 30°C for 3 hours. Reactions were stopped by addition of SDS sample buffer and boiling for 5 minutes at 100 °C.

The pSer incorporation was determined using the Phosphoprotein Phosphate Estimation Assay Kit (Thermo Scientific). The manufacturer’s protocol was modified by adding 0.2% polyvinyl alcohol (PVA) to minimize the precipitation of the malachite green-phosphomolybdate complex [17 (link), 18 ]. Briefly, 52 μg wt or pSer1412 nNOS and the phosvitin standard were diluted with 0.2 mL of TBS, incubated with 0.2 mL of 2.0 N NaOH at 65 °C for 30 min, and centrifuged at 12,000 g for 3 min after adding 0.2 mL of 4.7 N HCl. The supernatant was transferred into a new tube, and PVA was added to a final concentration of 0.2 %. After 30 min incubation at room temperature in dark, the absorbances of each standard and sample at 625 nm were measured on a Cary 50 UV-vis spectrophotometer. Lambda Protein Phosphatase (Lambda PP, New England Biolabs) was further used as a negative control to dephosphorylate both the wt and pSer1412 nNOSs: the wt and pSer1412 nNOSs were each incubated with 1000 units Lambda PP in 1X Reaction Buffer with added 1 mM MnCl2 at 30 °C for 30 min. After centrifugation in a 100 K cutoff centrifuge tube, the sample was exchanged into TBS for phosphate estimation assay. The obtained number of phosphate per nNOS subunit n is listed in Table S1.