The next day, filtrates were collected following centrifugation at 872 g, followed by 2 washes with 2.5 mL 0.5 M NaCl. survival (14). However, the precise mechanism by which ROS cause increased ABL1 activation remained undefined. We hypothesized that the increased ROS in HLRCC-associated PRCC might induce oxidation and inactivation of the PTP that normally dephosphorylates ABL1. Several methods for detecting PTP oxidation have been developed, including the in-gel PTPase assay, alkylating agent-based assays, dimedone-based assays, and specific antibodies against oxidized forms CH-223191 of PTPs (15). Nevertheless, identifying endogenously oxidized cellular PTPs remains challenging. Previously, we developed a mass spectrometry (MS)-based method, q-oxPTPome, that can monitor the oxidation CH-223191 of all catalytically active classical PTPs (16). We substantially refined this protocol, combining it with filter-aided sample preparation (FASP) (17), and increased its sensitivity by at least 10-fold. Using this approach, we found that several PTPs were highly oxidized in FH-deficient PRCC cell lines, and identified PTPN12 oxidation as the cause of increased ABL1 tyrosyl phosphorylation, establishing a novel mechanism of tumorigenesis. Materials and Methods Cell Lines and Cell Culture HEK293 T-REx cells were obtained from ThermoFisher. HeLa T-REx cells (early passage) were kindly provided by Dr. Brian Raught (Princess Margaret Cancer Centre, Canada). UOK262 FH-WT, UOK262 FH-deficient, UOK268 FH-WT and UOK268 FH-deficient cell lines (early passages) were generated in the Linehan laboratory. YUNK1 and YUNK1 were from Dharmacon. For immunoprecipitation, lysates (1C2 mg protein) were incubated with anti-Flag M2 affinity gel (Sigma) overnight at 4C, washed 4 in lysis buffer, resolved by SDS-PAGE and analyzed by immunoblotting. q-oxPTPome A buffer containing 20 mM Tris (pH 7.35), 150 mM NaCl, 10% glycerol, 1% NP40 was degassed overnight and then transferred to a hypoxia workstation (Don Whitley Scientific). Immediately before use, 30 mM N-ethylmaleimide (NEM; Sigma), 0.5 U/L catalase (Calbiochem), 0.125 U/L superoxide dismutase (Sigma) and a protease inhibitor cocktail (2 g/mL antipain, 2 g/mL pepstatin A, 20 g/mL leupeptin, and 20 g/mL aprotinin) were added to the lysis buffer. Within the workstation, cells were quickly washed once with degassed PBS, immediately lysed, incubated for 1 hr at 4C in the dark with constant rotation, and clarified at 10,000 g for 1 min at 4C. Supernatants were collected in the hypoxia workstation and buffer-exchanged by gel filtration (GE Healthcare) into degassed solution (20 mM HEPES [pH 7.4], 100 mM NaCl, 10% glycerol, Mouse monoclonal to CHUK 0.05% NP40, 0.02 U/L catalase). Desalted lysates were treated with 5 mM DTT and rotated in the dark for 1.5 hrs at 37C. Within the workstation, lysates were again buffer-exchanged to remove DTT, freshly prepared pervanadate was added to 8.5 mM, samples were rotated overnight at 4C in the dark and the next day, 4 mM EDTA was added. For immunoprecipitations, proteins were denatured in 0.5% SDS at 95C for 10 min, placed at RT for at least 30 min, and then diluted 5-fold in 50 mM CH-223191 HEPES (pH 7.4), 100 mM NaCl, 10% glycerol, 1% NP40 and 2 mM EDTA. Diluted samples were incubated over night at 4C with oxPTP antibody (5 g antibody/mg protein), and the next day, 40 L of protein G Sepharose (GE Healthcare) was added for another 3 hrs at 4C. For MS, proteins were incubated in 9 M urea with 4.5 mM DTT (Sigma) at 56C for 30 min, cooled to RT, and then treated with 10 mM iodoacetamide (IAM) for 20 min in the dark. Samples were then loaded into a 30K Centriprep (Millipore) pre-cleaned with 50 mM ammonium bicarbonate, centrifuged at 872 g for 15 min at RT, washed 3 with 50 mM ammonium bicarbonate (pH 8C8.5), and digested with trypsin (Promega) within the Centriprep overnight at 37C. The next day, filtrates were collected following centrifugation at 872 g, followed by 2 washes with 2.5 mL 0.5 M NaCl. Combined filtrates were acidified with trifluoroacetic acid (final pH~2) and desalted with Sep-Pak C18 Cartridges (Waters), following a manufacturers instructions. Desalted solutions were dried inside a SpeedVac, resuspended in 1 mL 50 mM HEPES (pH 7.4)/50 mM NaCl, incubated for 30 min on a shaker at RT, and centrifuged at 10,000 g for 5 min. After pre-clearing with 40 L of CH-223191 protein G Sepharose for 1 hr at 4C, samples were incubated with oxPTP antibody over night at 4C, and then 50 L of protein G Sepharose was added.
- Immune checkpoint inhibitors prevent malignancy cells from activating mechanisms that enable the cells to evade the hosts immune system
- One-tailed statistical analyses were performed in mouse experiments addressing the efficacy of a treatment in reducing metastatic burden
- When continue with clinical studies for mixture therapy, it’ll be imperative to use DNA-PK inhibitors in conjunction with DNA-damaging agents which have single agent efficacy, before trying combination therapy
- The former were designated as the continuous egg production hen (CEP) group and the last mentioned as the inactive egg production hen (IEP) group
- Hello world! on