Protein a g coated sepharose beads

Mouse monoclonal antibody against oxidized PTP active site (R&D Systems) was cross-linked to protein A/G-coated sepharose beads (Santa Cruz Biotechnology) with 2.5 mM disuccinimidyl suberate (Thermo Fisher Scientific) to prevent antibody co-elution. Upon elution of PTP motif peptides, antibody-crosslinked beads were regenerated for sequential immunoprecipitations from the same sample, by re-using the flowthrough. For PTP motif peptide immunoprecipitations, fin lysates were digested sequentially at 37 °C in Lys C (1:100) and Trypsin (1:50) for 4 and 12 hours respectively, and diluted in 50 mM HEPES, pH 7.4. PTP motif peptides isolated from 5 sequential immunoprecipitations were further purified by strong cation exchange STAGE tips and analysed individually by LC-MS/MS as 5 technical replicates.

An MTT assay kit was purchased from Roche. A site-directed mutagenesis kit was purchased from Stratagene. 6-Hydroxydopamine hydrobromide (6-OHDA), DMSO, SB216763 (S3442, an inhibitor of GSK3β) and AGK2 (A8231, an inhibitor of SIRT2) were obtained from Sigma-Aldrich. Antibodies against pGSK3β (Ser9) and GSK3β were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against SIRT2, ace-tubulin, α-tubulin, HA and Flag were purchased from Sigma-Aldrich. Secondary antibodies conjugated to Alexa 488 or Alexa 594 were purchased from Invitrogen. Hoechst 33258 (94403) was purchased from Sigma-Aldrich. Protein A/G-coated Sepharose beads were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). An anti-phosphoserine/threonine/tyrosine antibody was obtained from Abcam (ab15556). Protein kinase CDK5/p25 (cat. 14—516) and GSK3β (cat. 14—306) were purchased from Millipore. Cells were transfected using Lipofectamine 2000 Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Other chemicals and reagents were of the highest analytical grade and were purchased from local commercial sources.