Phosphatase inhibitors

The mass spectrometry assay was performed as described previously [23 (link)]. Briefly, HEK293T cells were transfected with N-terminally Flag-tagged LRP6-ΔN expression vector or empty vector. The total protein of the cells was harvested with RIPA buffer containing 50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, protease inhibitors (Bimake, Beijing, China), and phosphatase inhibitors (Topscience, Shanghai, China), followed by centrifugation. Immunoprecipitations were performed using anti-Flag agarose beads (Bimake, Beijing, China) incubated with the supernatant overnight at 4 °C. The beads were washed three times with RIPA buffer. Beads with extracted proteins were digested by trypsin (Promega, Shanghai, China). The peptide sequences from these proteins were then extracted for mass spectrometry analysis on a Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA).

Flag-tagged PTPN21 (1 μg/μl; WT or mutant) plasmids alone or in the presence of HA-tagged 18E7 plasmids (0.25 μg/μl) were tranfected into HEK293T cells. Forty-eight hours after transfection, cells were washed twice with ice-cold PBS and lysed in 250 μl of ice-cold lysis buffer [20 mM tris-HCl (pH 8.0), 0.15 M NaCl, 10% glycerol, and 0.5% NP-40], supplemented with EDTA-free protease inhibitors (Topscience, catalog no. C0001), phosphatase inhibitors (Topscience, catalog no. C0004), and UltraNuclease (Yeasen Biotechnology, catalog no. 20156ES). Cell lysates were centrifuged at 700g for 5 min, and the supernatants were incubated with Flag beads at 4°C for 4 hours. The beads were washed three times with lysis buffer, mixed with 1× sample loading buffer, and boiled at 95°C for 5 min for Western blotting.