Bxp 53

Tissue samples of the motor cortex and SC (control n = 6, sporadic [sALS] n = 6, and familial [fALS], n = 5) were homogenized in lysis buffer (20 mM TRIS-HCl [pH 8], 137 mM NaCl, 1 mM Na₃VO₄, 8% glycerol, 1% triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 10 µg/mL typsin inhibitor). Next, they were centrifuged for 15 minutes at 18 000g. Protein content was determined using the Bio-Rad (Hercules, CA) DC protein assay kit. Per sample, 40 µg of proteins was loaded on a NuPAGE 4%–12% Bis-Tris Midi protein gels, 26-well (Thermo Fisher Scientific, Waltham, MA) and electrophoreses was performed for 2 hours at 30 mA. The temperature of the running buffer (MOPS buffer [Invitrogen, Carlsbad, CA] 1:20) was maintained below 10°C. Gels were dry-blotted on nitrocellulose and these were probed for P-gp with MDR1/ABCB1(E1Y7S) rabbit mAb #13978 1:1000 (Cell Signaling Technologies, Leiden, the Netherlands) or for BCRP with BXP-53 1:400 (ab24115; Abcam). Secondary HRP-antirabbit or HRP antirat 1:2000 (both from DAKO, Santa Clara, CA), respectively, were used and the blot exposed to ECL reagent (35 µg/mL coumaric acid, 220 µg/mL luminol, 0.0075% H₂O₂ in 100 mM TRIS pH 8.5) and images were taken on a Chemidoc XRS+ (Bio-Rad) and analyzed using Image Lab software (v5.2.1).