Novex nupage sds page gels

Cell lysates obtained as described above were mixed with SDS electrophoresis sample buffer containing 90 mM DDT, boiled and subjected to electrophoresis in 4–12% gradient Novex NuPAGE SDS-PAGE Gels (Invitrogen). Resolved proteins were transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Etobicoke, ON, Canada), and membranes were blocked in 5% skim milk and probed with various antibodies. Primary antibodies were monoclonal α-NS1 [42 (link)], monoclonal α-NP [44 (link)] (gift from Dr. Mingyi Li, National Microbiology Labs), monoclonal α-M1 (Thermo Fisher, MA1-80736), polyclonal α-M2 (Thermo Fisher, PA5-32233), monoclonal α-beta-actin (Cell Signalling, 3700S, Danvers, MA, USA), α-NUMA1 (Bethyl Laboratory, A301-510A, Montgomery, TX, USA), α-PRPF19 (Bethyl Laboratory, A300-101A) and/or α-UTP6 (Thermo Fisher, PA5-21716). Primary antibodies were detected with HRP-linked polyclonal α-mouse (Cell Signalling, 7076S), polyclonal α-rabbit (Cell Signalling, 7074S) or monoclonal VeriBlot™ (Abcam, Ab131366, Toronto, ON, Canada) secondary antibodies and HRP signals were detected using enhanced chemiluminescence (ECL) reagent (prepared in house). Images were taken with an Alpha Innotech Fluor Chem^® Q Imaging System and minimally processed by Adobe® Photoshop^® software (San Jose, CA, USA).

Control and PARK2 post-mortem brain tissues were from the Department of Neurology, Juntendo University School of Medicine. The study protocol was approved by the Human Ethics Review Committee at Juntendo University School of Medicine. Informed consent to use human tissues were obtained from patients or close family members. Patients’ data are reported in Supplementary Table 1. Control brain tissues were autopsied brain tissue from age-matched controls who had undergone neuropathological examination to exclude neurodegenerative disorders. Tissues were homogenized with lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitors (Roche), 50 μM MG132 and 10 mM N-ethylmaleimide (Sigma)). Western blottings were performed with Novex NuPAGE SDS–PAGE gels (Invitrogen). The following antibodies were used: GluA1 ((13185, Cell Signaling, 1:500), GluA2/3 (AB1506, Millipore, 1:1,000), GluN1 (clone N308/48, NeuroMab, 1:300), GluN2B (PA5-18536, Thermo Scientific, 1:300), GluK1 and GluK3 (H0002897 and H0002899, respectively, Abnova, 1:2,000), GluK2 (TA310550, Origene, 1:1,000), parkin (P6248, Sigma, 1:6,000), GAPDH (sc-25778, Santa Cruz, 1:1,000).

Parkin-Q311X (A) mice and littermate controls (all mice were female FVB/N strain) were purchased from Charles River (Calco, Italy). Mice were kept under environmentally controlled conditions on a 12-h light/dark cycle with food and water ad libitum. Three- to four-week-old mice were killed and the substantia nigra were immediately dissected out on ice. Tissues were homogenized with lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitors (Roche), 50 μM MG132 and 10 mM N-ethylmaleimide (Sigma)). Western blottings were performed with Novex NuPAGE SDS–PAGE gels (Invitrogen). The following antibodies were used: GluA1 (13185, Cell Signaling, 1:500), GluA2/3 (AB1506, Millipore, 1:1,000), GluN2B (PA5-18536, Thermo Scientific, 1:300), GluK2 (TA310550, Origene,1:1,000) and GluK3 (H0002899, Abnova, 1:2,000), GAPDH (sc-25778, Santa Cruz, 1:1,000), Calcineurin A (ADI-SPA-610, Enzo Life Sciences 1:1,000) and Spectrin (2122, Cell Signaling, 1:1,000). The study was approved by The Ethics Committees of IRCCS Istituto Auxologico Italiano ``Comitato Etico dell'IRCCS Istituto Auxologico Italiano di Milano''. Experimental procedures took place in accordance with the European Communities Council Directive (86/809/EEC).