Af3865

Proteins were separated with 4 to 12% bis–tris gels for eEF2 or 7% tris–acetate gel for TNR and transferred to a PVDF membrane. Membranes were blocked and incubated overnight with primary antibodies against pT57-eEF2 (1/1000, 2331, Cell Signaling Technologies), eEF2 (1/1000, 2332, Cell Signaling Technologies), TNR (1/2000, AF3865, R&D systems), alpha-tubulin (1/10,000, ab7291, Abcam), or SPP1 (1/1000, ab8448, Abcam). Membranes were washed and incubated with HRP conjugated secondary antibodies against rabbit IgG (1/10,000, 711-035-152, Jackson Immunoresearch), goat IgG (1/10,000, 705-035-003, Jackson Immunoresearch), or mouse IgG (1/10,000, ab6789, Abcam). Chemiluminescence was detected using ECL substrate and imaged with FlourChem M (Biotechne) or Amersham Imager 600 (GE Healthcare Life Sciences) for membranes from 2D gel. For detection of total eEF2 after detection of p-eEF2, the membrane was stripped with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific). Quantification was performed using ImageJ software. Contrast was adjusted using adobe illustrator to improve visualization.

Formalin fixed paraffin embedded DRG tissue sections were deparaffinized and subject to citrate buffered antigen retrieval. Sections were stained using a primary antibody raised against TNR (1/50, AF3865, R&D systems) and secondary antibody conjugated to Rhodamine Red-X raised against goat IgG (1/100, 705-296-147, Jackson Immunoresearch). Sections were also stained with antibody raised against NeuN and conjugated to Alexa Flour488 (1/100, MAB377X, Sigma Aldrich). Sections were mounted using DAPI fluoromount-G (17984-24, Electron Microscopy Sciences). Fluorescence was imaged using a Nikon A1R HD25 Spectral microscope. Representative images at 10× magnification were used for analysis, images for figures were acquired at 20× magnification. The proportion of NeuN expressing cells which are also surrounded by TNR was compared between groups.