Odyssey lc scanner

Mouse brain samples were extracted using the same method described above for the MS analysis. For each analysis, 5–20 μg of sample was added per lane, separated on 12% Bis-Tris Pre-Cast gels (Thermo Fisher) and transferred to PVDF membranes using 7.5% BSA in TBS for blocking. All primary antibodies were used overnight at 1:1000. Antibodies were used against TH (tyrosine hydroxylase; EMD Millipore, 657012 RRID:AB_696697), DAT (dopamine transporter; EMD Millipore, MAB369 RRID:AB_2190413), Ddc (aromatic-L-amino-acid decarboxylase; Abcam, ab3905 RRID:AB_304145), NSE (neuron specific enolase; Abcam, ab53025 RRID:AB_881756), PKC-β2 (protein kinase C beta-2; Abcam, ab32026 RRID:AB_779042), Akt (protein kinase B; Cell Signaling, 9272 RRID:AB_329827), Lmp7 (proteasome subunit beta type-8; Abcam, ab3329 RRID:AB_303708), and α-synuclein clone D37A6 (Cell Signaling Technology, #4179 RRID:AB_1904156) or clone Syn211 (Sigma-Aldrich Cat# S5566, RRID:AB_261518). Antigen-antibody complexes were detected using an Odyssey LC scanner (LiCor) after incubation with appropriate secondary antibodies. Densitometry was used to quantify intensity of protein bands.

U-251 cells were grown in high glucose DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, 1% glutamine and 1% HEPES. Cells were plated at low density and allowed to adhere for 24 h, after which cells were pretreated with 0.1 μg/mL human interferon gamma (Pepro Tech Inc.) alone or in conjunction with the Lmp7 inhibitor ONX 0914 (Cayman Chemicals) at a final concentration of 0.2 μM. Following incubation overnight, 5 μg/mL α-synuclein fibrils were added to the media and incubated for 4d. The cells were washed 3× with PBS and lysed in PBS + 1% Trition-X100. 12 μg of protein was loaded into each lane and the western proceeded as described above. Primary antibodies to α-synuclein (clone Syn211, SigmaAldrich, S5566 RRID:AB_261518), Lmp7 (proteasome subunit beta type-8; Abcam, ab3329), and Actin (Sigma Aldrich, A2066 RRID:AB_476693) were incubated with the membrane at a concentration of 1:1000 overnight at 4 °C (Syn211) or for 1 h at room temp (Lmp7, Actin). IR Dye conjugated anti-mouse or anti-rabbit secondary was incubated at 1:5000 for 1 h and the blots were visualized using an Odyssey LC scanner (LiCor).