Tyrosine phosphatase assay system

Phosphatase activity of the SCI site from eight- to ten-week-old rats with either SCI or conditioning lesion (n = 3 per condition) was assayed in 96-well microtiter plates using a tyrosine phosphatase assay system (Promega, Carnaxide, Portugal). Protein extracts (25 μg) were incubated with 100 μM of a P-GSK3βTyr216 peptide (Abcam, Cambridge, UK) at 30°C for 30 minutes. The released phosphate was determined by measuring the absorbance at 620 nm. Control reactions without the P-GSK3βTyr216 peptide were performed.

PTP1B phosphatase activity assay was performed using a tyrosine phosphatase assay system (Promega). Endogenous phosphates were removed from the tissue extracts or cell lysates using sephadex spin column which were subsequently incubated and immunoprecipitated with an antibody targeting PTP1BA reaction mix was then prepared using a tyrosine phosphopeptide substrate (ENDp(Y)INASL). Immunoprecipitated protein (2 μg) was incubated with the substrate for 20 min at 37 °C in a 96-well plate. The reaction was terminated using molybdate dye/additive mixture for 15 min at room temperature. Absorbance of the samples was measured using a plate reader at 630 nm. Phosphatase activity was calculated using the phosphatase assay standard supplied with the kit.

Freshly enriched NK cells were lysed using 25 mM imidazole HCl (pH 7.0), 1 mg/ml BSA, 1% Triton X-100, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride. Protein tyrosine phosphatase activity of murine NK cell lysate was measured using a kit from the Tyrosine Phosphatase Assay System (Promega). Briefly, endogenous phosphates were removed from cell lysate by passing through Sephadex columns twice. 250,000 NK cells were used per reaction, and each reaction was performed in duplicates. Phosphate-free lysate was incubated with indicated concentrations of H₂O₂ for 1 h at 37°C in the presence of phosphorylated peptides provided by the kit. The reactions were stopped using the molybdate dye, and the colour was developed at RT for 15 min. The amount of molybdate–phosphate complex was measured at 590 nm absorbance using the Infinite 200 Pro plate reader (Tecan).

PTP activity was measured using a Tyrosine Phosphatase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. NCI-H1703 cells were transfected with siDDIAS for 72 h and lysed with storage buffer. Spin columns were used to remove endogenous free phosphate from the cell lysates. Tyrosine phosphatase activity was measured using the EMax plate reader system (Molecular Devices, San Jose, CA, USA) at 600 nm.

Measurement of SHP-2 activity was done according to Godfrey et al.^{49 (link)}, with minor modifications. Steps to measure SHP-2 activity were performed on ice and inside of an anoxic chamber (GB2202-P-V, MBraun). The activity of SHP-2 was measured in immunoprecipitates using the Tyrosine Phosphatase Assay System (Promega). Detailed protocol can be found in the electronic supplementary information.