Sti571

The pMX-INV or pMX-DEL^CJ substrate was introduced in pro-B cell lines through retroviral infection and cells that had integrated the recombination substrate were enriched based on hCD4 expression7 (link)34 (link). For V(D)J recombination assay, v-abl transformed Bcl2/pMX-INV infected pro-B cells (10⁶ per ml) were treated with 3 μM of the v-abl kinase inhibitor STI571 (Novartis) or 0.3 μM of the STI571-analogous v-abl kinase inhibitor PD180970 (Sigma) and assayed for rearrangement by FACS analysis of GFP expression or Southern blotting at 0, 72 or 96 h. In some experiments, the ATM kinase inhibitor KU55933 was added at 15 μM together with PD180970 or STI571 (named ABLki). For FACS analysis, V(D)J recombination efficiency was scored as the percentage of GFP positive cells among hCD4-positive cells (human CD4-PE, Miltenyi, 1:20 dilution). For ABLki release experiments, cells were collected, washed and cultured without ABLki for 3–4 days before metaphases preparation.

Cell lines used in this study were murine neuroblastoma cells N2a (obtained from ATCC; CCL-131) and neuronal CAD5 [54 (link)] cells, respectively, not infected or permanently infected with 22L or RML prions, and mouse embryonic fibroblast (MEF) cells uninfected or persistently infected with ME7 prions. N2a-wt cells [9 (link)] are N2a stably overexpressing murine PrP^C, and 3F4-N2a and RML-N2a [55 (link)] cells stably overexpress murine PrP containing the epitope for mAb 3F4 and were established by our group. N2a cells were cultured in Opti-MEM (Invitrogen) containing 10% (v/v) fetal bovine serum (FBS; PAA) and penicillin/streptomycin. CAD5 cells were grown in Opti-MEM with 10% bovine growth serum (Hyclone). MEF cells were kept in MEM (Invitrogen) with the addition of 10% (v/v) fetal bovine serum and penicillin/streptomycin. All cells were grown at 37 °C in a 5% CO₂ atmosphere. Purified PAs were added to the culture media at different concentrations and various durations. Fresh PAs were added with each media change which was done every other day. When indicated 10 mM NH₄Cl was added to the culture media 24 h before lysis. STI571 (Sigma Aldrich) was dissolved in DMSO at a stock concentration of 10 mM.

HEK293 cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) Fetal Bovine serum (GE Healthcare, Little Chalfont, UK), 2 mm glutaMAX (ThermoFisher Scientific, Waltham, MA, USA), 100U·mL⁻¹ Penicillin and 100 μg·mL⁻¹ Streptomycin (ThermoFisher Scientific). Anti‐GST, Anti‐FLAG M2 antibody, HA antibody and agarose resins were purchased from Sigma (St. Louis, MO, USA). Glutathione sepharose 4B beads were from GE healthcare. Anti‐phosphotyrosine antibody (4G10) was from Millipore (Billerica, MA, USA), PKD anti‐pSer‐744/748 antibody, anti‐PKCδ antibody, secondary HRP‐linked goat anti‐Rabbit and Horse, anti‐Mouse antibodies were from Cell Signaling Technologies (Beverly, MA, USA). An in‐house site‐specific phospho‐Tyr antibody targeting pTyr in the P + 1 loop (CPApYLAPEV), which is cross‐reactive between PKD isoforms due to 100% homology of the epitope is described previously 36. Phorbol 12,13‐dibutyrate (PDB), ATP, STI‐571, PP2, CRT 0066101, CID 755673 and Hydrogen peroxide 30% (v/v) were from Sigma, Polyethyleneimine (PEI) was from Polysciences Inc. (Warrington, PA, USA).