Hepes buffered saline

For the CGRP release assay, DRGs were dissected from C57Bl6/J mice into Hank’s balanced salt solution, incubated for 90 min at 37°C with 1 mg/ml collagenase A and 2.4 U /ml dispase II (Roche Applied Sciences) in HEPES-buffered saline (Sigma), and then mechanically triturated using glass Pasteur pipettes. DRGs were then centrifuged over a 10% BSA gradient and plated on laminin-coated 96 well plates at approximately 5,000 cells per well. A plate was prepared from each of two mice, each plate with DRGs in 10 wells. DRGs were cultured 48 hr before conducting a CGRP release assay in B27-supplemented neurobasal-A medium plus 50 ng/ml NGF, 2 ng/ml GDNF, 10 µM arabinocytidine and penicillin/streptomycin. The DRG neurons were exposed to 50 mM KCl with vehicle (2 wells of each plate), 50 mM KCl in the presence of 30 µM NNCB-2 (two wells/plate), or 50 mM KCl in the presence of 30 µM CNCB-2 (four wells/plate), with two wells/plate serving as controls with no KCl depolarization (4 mM KCl), for 10 min at 37°C. The supernatants were collected and analyzed using the Rat CGRP Enzyme Immunoassay Kit (Bertin Pharma/Cayman Chemical, #589001). Plates were read at 405 nm for 0.1 s on a Wallac Victor 1420 Multilabel Counter (Perkin Elmer) and data was analyzed using GraphPad Prism. Data are shown as raw values of absorbance and bars and error bars indicate mean ± SEM.

To generate stable transfectants expressing Zap70^wt or Zap70^C564A, P116 cells were transfected with 5–30 µg of pPB[Exp]-Puro-EF1A>hZAP70/T2A/EGFP and 5 μg of pEF_hyPBase. For transient transfections of P116 cells, we used 5–30 µg of the pEYFP-N1-hZap70 vector. DNA electroporation of P116 cells was performed using the Gene Pulser II System (BIORAD) as previously described [16 (link)]. The transfected cells were cultured in an RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. A total of 0.5 μg/mL of puromycin (Gibco, Waltham, MA, USA) was added for the generation of stable transfectants, which were additionally sorted with the Aria Cell Sorter 3 (BD Bioscience) and afterwards maintained in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.1% Ciprobay.
For the transfection of HEK293T cells, 1 × 10⁶ cells were seeded onto 6-well tissue culture plates one day before transfection. Separately, a mixture was prepared containing 300 μL of 250 mM CaCl₂ and 10 μg of pEYFP-N1-hZap70, which was added dropwise under constant agitation to 300 μL of HEPES buffered saline (Sigma-Aldrich). After incubation for 45 min at RT, 3 mL of DMEM (PAN Biotech, Aidenbach, Germany) were added to the mixture, which was subsequently carefully pipetted into the well containing the cells. Cells were incubated in the presence of a transfection medium for 24 h.

Lentivector particles were produced using the CaCl₂ method based by tri-transfection with the plasmids pCMV-dR8.91 and pCMV-VSVG, CaCl₂ and Hepes Buffered Saline (Sigma-Aldrich, Saint-Quentin-Fallavier, France), into HEK-293FT cells. Viral supernatants were harvested 48 hr after transfection, passed through 0.45 μm PVDF filters (Dominique Dutscher SAS, Brumath, France), and stored in aliquots at –80 °C until use. Viral production titers were assessed on HT1080 cells with serial dilutions of a lentivector expressing GFP and scored for green fluorescent protein (GFP) expression by flow cytometry analysis on a BD FACSVerse (BD Biosciences, Le Pont de Claix, France).

The 12F6 antibody was prepared and characterized as previously described^{27 (link),32 (link),45} . Bovine serum albumin (BSA), goat anti-mouse IgG polyclonal antibody, tetrachloroauric acid (HAuCl₄), trisodium citrate, Tween 20, sucrose and the reagents used to prepare HEPES-buffered saline (HBS: NaCl 137 mM, KCl 3 mM and HEPES 10 mM; pH 7.4) were purchased from Sigma Aldrich. Analytical grade nitric acid was purchased from Fisher Scientific. The U(VI) stock solution was purchased from Perkin Elmer and the chelator, 2,9-dicarboxyl-1,10-phenanthroline (DCP) was purchased from Alpha Aesar. Cellulose membranes (CFSP001700), glass fiber (GFCP00080000), nitrocellulose membranes (HF180), and adhesive laminated cards were purchased from Millipore.

Cells were treated with 50 mM etomoxir and/or 200 nM INK128 for 48 h as described in Results. Cells were trypsinized and plated on Matrigel (Sigma-Aldrich, CLS356237) pre-coated Ibidi µ-Slide 8 Well slides (Ibidi, 80821). After 30 min of attachment, cells were stained for 1 h with SPY650-DNA (SpiroChrome, SC501) in culture medium with inhibitors at 37 °C, washed twice with HEPES-buffered saline (Sigma-Aldrich, 40010) and stained for 2 h with 20 µM FAOBlue (DiagnoCine, FNK-FDV-0033) in DMEM with inhibitors at 37 °C in 20% O₂ and 5% CO₂. Cells were washed once with HEPES-buffered saline and imaged with 200 µl HEPES-buffered saline. Imaging was done on a Zeiss Plan-Apochromat 20×/0.8 objective on the Zeiss LSM880 Airy microscope using longitudinal-section magnetic mode. Images were processed using Fiji ImageJ2 (version 2.3.0) and quantified using CellProfiler (version 4.2.1).