Molecular weight cutoff spin filters

HPLC was conducted on plasma and hippocampal, CeA, and DR tissue homogenates as previously published (Finnell et al., 2017a (link); Finnell et al., 2017b (link)). Briefly, all samples were spiked with 10nM dihydroxybenzlamine (DHBA, Thermo Fisher Scientific, Waltham, MD) to serve as an internal standard and filtered via centrifugation using Amicon Ultra 0.5 mL Ultracel 3kDa (tissue homogenates) or 50kDA (plasma) molecular weight cut off spin filters (Millipore, Billerica, MA) for 30 min at 4°C and 16,000xg. Flow through collected from the spin filters were used for HPLC analysis. All NE data were normalized to DHBA and tissue homogenates were further normalized to BCA protein concentrations. The hippocampus served as the NE lesion verification site as it exclusively receives NE innervation from the LC (Loy et al., 1980 (link)) and confirmed that DSP-4 treatment reduced NE by approximately 85% (Average Hippocampal NE pmol/ug protein ± SEM; Vehicle: 34.54 ± 6.4; DSP-4: 4.9 ± 1.5; t₍₁₁₎=4.2, p<0.01). These data are comparable to studies using the standard 50 mg/kg intraperitoneal dose (Cheetham et al., 1996 (link); Harro et al., 1999 (link); Harro et al., 1995 (link); Kask et al., 2000 (link)) and direct ICV injection of 400µg of DSP-4 (Sziray et al., 2010 (link)) in which hippocampal NE was depleted by 70–100%.

Recombinant toyocamycin nitrile hydratase was expressed and purified as described previously^{24 (link)}. Rabbit pyruvate kinase was purchased from Roche; the phosphoglycerate mutase 2 was discovered as a contaminant in that sample. Glyceraldehyde 3-phosphate dehydrogenase was isolated from HeLa S3 cells as described previously²⁵ . Ferritin from horse spleen was purchased from Sigma Aldrich. Finally, human 20S proteasome was purchased from Enzo Life Sciences. All samples were desalted prior to analysis by buffer exchanging with molecular weight cutoff spin filters (Millipore) into 150 mM ammonium acetate at pH 7.