Anti gadd34

Postmortem brain samples from neuropathologically confirmed PD cases (n=5) and age- and gender-matched controls (n=7) were obtained from the New York Brain Bank at Columbia University (New York, NY). Midbrain sections (6 µm) were deparaffinized in xylene and rehydrated in an ethanol series. Sections were then cooked for antigen retrieval in citrate buffer for 45 min at 100°C. Sections were then blocked in goat serum for 20 min and incubated with anti-GADD34 (Proteintech) at 1:200 in blocking buffer overnight at 4°C. To test the specificity of the antibody, some sections were incubated with the antibody that was mixed with GADD34 fusion protein (Proteintech) Sections were then washed and incubated with biotinylated anti-rabbit secondary antibody for 1 h at room temperature, washed, and incubated in ready-to use ABC complex solution (Vectastain, Vector Laboratories) at room temperature, and then SG substrate (Vector Laboratories) was added and left on the sides for 15 min. Sections were counterstained with Nuclear Fast Red, dehydrated and mounted with coverslips. For quantification of GADD34 staining pattern, neuromelanin-positive neurons were assessed for the presence of GADD34-positive granules in a blinded manner. At least 30 neurons were assessed for each case.

Stock solutions of 6-hydroxydopamine (6-OHDA; Tocris), guanabenz acetate salt, clonidine, efaroxan (Sigma) were prepared in ddH2O; camptothecin (CPT; Tocris) was prepared in DMSO. The following antibodies were used: anti-ATF4 was commercially generated for our laboratory (Liu et al., 2014 (link); Pasini et al., 2015 (link)); anti-GADD34 was from Proteintech; anti-tyrosine hydroxylase (TH) was from EMD Millipore; anti-ERK was from Santa Cruz Biotechnology; anti-parkin (PARK8), anti-phospho-eIF2α (S51) and total eIF-2α were from Cell Signaling Technology.