Anti frataxin

After RNA extraction with Qiazol, the proteins were precipitated with isopropanol at room temperature (RT) and washed with 0.3 M guanidine hydrochloride and pure ethanol. A resuspension buffer (4% SDS, 0.025 M Tris HCl, 7.5% glycerol, 0.5% β-mercaptoethanol, and bromophenol blue) was used for protein denaturation at 100°C for 5 min. The concentration of proteins was quantified with the Amido black test.⁶⁴ (link) Then 25–40 μg protein was separated on a 15% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Mississauga, ON, Canada).
After blocking, various primary and secondary antibodies were used as follows: anti-frataxin (ab110328, Abcam, Toronto, Canada), anti-GAPDH (Millipore Sigma MAB374, Etobicoke, ON, Canada), and anti-PGC-1α and anti-GAD65 (Thermo Fisher Scientific PA5-38022 and PA5-22260, Rockford, IL, USA). The secondary antibody used was a rabbit anti-mouse and anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA). After 3 washes in 0.1% PBS and 0.05% Tween for 10 min each, the membrane was revealed using the Clarity Western ECL Substrate kit (Bio-Rad, Mississauga, ON, Canada) and developed to visualize the protein bands. These were quantified with the ImageJ software.

Cells were treated with 100 nM BIX01294, 2 μM GSK126 or combination of both for 72 h. Cells were washed with ice-cold PBS and lysed on ice for 30 min with cell lysis buffer (Cell Signaling Technology, 9803) supplemented with 400 μM PMSF protease inhibitor (Cell Signaling Technology, 8553). Lysates were subjected to centrifugation at 12,000 g for 30 min at 4°C and protein concentrations were determined using the BCA protein assay reagent (Thermo Scientific). 50 μg of protein lysates were boiled for 10 min and subjected to SDS-PAGE electrophoresis using 4–15% precast gels (Bio-Rad, 567-1084). Densitometry was calculated using the Image Lab Software 5.2.1 (Bio-Rad). Antibodies used in this study are as follows: anti-frataxin (1:250, ab113691, Abcam), β-actin (1:1000, A2066, Sigma), goat anti-Rabbit HRP (1:5000, P0448, Dako) and goat anti-Mouse HRP (1:5000, P0447, Dako).