PKM2 is also the substrate of protein-tyrosine phosphatase 1B: inhibition of PTP1B increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity

PKM2 is also the substrate of protein-tyrosine phosphatase 1B: inhibition of PTP1B increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity. parkin or PKM2. After washing five instances with BC100 buffer (20 mm Tris-HCl, pH 7.9, 100 mm NaCl, 10 mm KCl, 1.5 mm MgCl2, SOS1-IN-1 20% glycerol, and 0.1% Triton X-100), the bound proteins were eluted by 1 SDS loading buffer with warmth to denature proteins. On the other hand, cell cytoplasmic components were incubated with FLAG-agarose beads (Sigma) or HA-agarose beads (Roche Applied Technology) at 4 C over night to analyze cells transfected with FLAG-tagged or HA-tagged plasmid. The beads SOS1-IN-1 were washed five instances with BC100 buffer, and the bound proteins were eluted using FLAG peptide or HA peptide in BC100 buffer for 2 h at 4 C. Protein Complex Purification Protein complex purification was performed as explained previously (30, 31) with some modifications. The cytoplasmic components of the FLAG-HA-parkin/H1299 stable lines or FLAG-HA-PKM2/H1299stable lines were prepared as explained above and subjected to a FLAG M2 and HA two-step immunoprecipitation. The tandem affinity-purified parkin or PKM2-connected proteins were analyzed by liquid chromatography (LC)-MS/MS. GST Pulldown Assay GST or GST-tagged fusion proteins were purified as explained previously (30, 31). [35S]Methionine-labeled proteins were prepared by translation using the TnT Coupled Reticulocyte Lysate System (Promega). GST or GST-tagged proteins were incubated with 35S-labeled proteins at 4 C over night in BC100 buffer + 0.2% BSA and then incubated with GST resins (Novagen) for 4 h. The resins were washed five instances with BC100 buffer. The bound proteins were eluted with 20 mm reduced glutathione (Sigma) in BC100 buffer for 2 h at 4 C and resolved by SDS-PAGE. The drawn down 35S-labeled protein was recognized by autoradiography. Parkin Knockdown Ablation of parkin was performed by transfecting cells with siRNA duplex oligonucleotides (On-Target-Plus Smart Pool: 1, catalog quantity J-003603-05; 2, catalog quantity J-3603-06; 3, catalog quantity J-3603-07; and 4, catalog quantity J-3603-08) from Thermo Sciences and control siRNA (On-Target-Plus-Si Control Nontargeting Pool, D00181010, Dharmacon). The cells were transfected three times. Ablation of parkin in MCF10A cells were performed by illness with shRNA lentivirus. Parkin-specific shRNA plasmids and control shRNA plasmid were received from Thermo Sciences (1, catalog quantity V2LHS_84518; 2, catalog quantity V2LHS_84520; 3, catalog quantity V3LHS_327550; and 4, catalog quantity V3LHS_327554). The lentivirus was packaged in 293T cells and infected cells as explained in the manufacturer’s protocol. Ablation of parkin in U87 cells and FLAG-HA-parkin/U87 stable collection was performed by transfecting cells once having a pool of four siRNA duplex oligonucleotides against parkin 3-UTR region (1, CCAACTATGCGTAAATCAA; 2, CCTTCTCTTAGGACAGTAA; 3, CCTTATGTTGACATGGATT; 4, GCCCAAAGCTCACATAGAA). Cell-based Ubiquitylation Assay The ubiquitylation assay was performed as explained previously (32) with some changes. 293 cells were transfected with plasmids expressing FLAG-PKM2, myc-parkin, and His-ubiquitin. After 24 h, 10% of cells were lysed with radioimmune precipitation assay buffer, and components were preserved as input. The rest of the cells were lysed with phosphate/guanidine buffer (6 m guanidine-HCl, 0.1 m Na2HPO4, 6.8 mm Na2H2PO4, 10 mm Tris-HCl, pH 8.0, 0.2% Triton X-100, and freshly added 10 mm -mercaptoethanol and 5 mm imidazole), sonicated, and subjected to Ni-NTA (Qiagen) pulldown overnight SOS1-IN-1 at 4 C. The Ni-NTA resin-bound proteins were washed with wash buffer 1 (8 m urea, 0.1 m Na2HPO4, 6.8 mm Na2H2PO4, 10 mm Tris-HCl, pH 8.0, 0.2% Triton X-100, and freshly added 10 mm -mercaptoethanol and 5 mm imidazole) once and further washed with wash buffer 2 (8 m urea, 18 mm Na2HPO4, 80 mm Na2H2PO4, 10 mm Tris-HCl, pH 6.3, 0.2% Triton X-100, and freshly added 10 mm -mercaptoethanol and 5 mm imidazole) three times. SOS1-IN-1 The bound proteins were eluted with elution buffer (0.5 m imidazole and 0.125 m DTT) and resolved by SDS-PAGE. TPO To purify ubiquitylated PKM2, 1st all His-ubiquitin-conjugated proteins including PKM2 were purified with Ni-NTA resin as explained above and eluted with elution buffer (0.5 m imidazole in BC100 buffer). The eluants were dialyzed with BC100 buffer for 16 h at 4 C, exchanging the buffer for new buffer five instances during that period. Then the eluants were incubated with the FLAG M2-agarose beads.