Ab65311

The antibodies used for western blotting and immunohistology in this study were as follows: COXIV (ab160561, Abcam), Tom40 (sc-365467, Santa Cruz Technology), PINK1 (D8G3, 6946, Cell Signaling Technology), LC3B (2775S, Cell Signaling Technology), p17 (Ribosomal Protein L29 [P-14], sc-103166 Santa Cruz Biotechnology), P-tau^Ser202^/Thr205 (AT8—MN1020 Thermofisher), P-tau^Thr231 (AT180—MN1040 Thermofisher), anti-Tau [TAU-5] (ab80579, Abcam), MBP (ab218011, Abcam), anti-β-actin-peroxidase antibody (A3854, Millipore Sigma), Cytochrome c (ab65311, abcam), and CerS1 (MBS2523738, MyBiosource).

Cells were washed in cold PBS, collected in 100-μl Cytosol Extraction Buffer containing DTT and protease inhibitors (ab65311, Abcam) and immediately homogenized using Branson Sonifier 150 on ice. Protein concentration was measured employing Pierce^TM BCA protein Assay Kit (23225, Thermo Scientific) on a Nanodrop 2000 Spectrophotometer (Thermo Scientific). Ten-microgram protein aliquots were loaded on a 12% SDS-PAGE gel, and western blot was performed using the following manufacturer instructions. After membrane develop, the films were scanned (MC332, OKI), and images were analyzed using the Image J 1.42q (National Institutes of Health, USA). Briefly, images were converted to grayscale 8 bit. Around the first lane, a rectangle was drawn using the rectangular selection tool. This first line, corresponding to the control group, served as a reference. Using the function Analyze>Gels>Plots Lanes, the intensity of every other lane in the image was analyzed. Results were expressed as percentage of the total size of all of highlighted peaks. Values were pasted into Excel for further analysis.

Immunofluorescence: Mouse anti-cytochrome c (1:100 dilution, ab65311) and rabbit anti-desmin (1:80 dilution, ab8592) were from Abcam (Cambridge, UK). Mouse anti-α-tubulin (1:2000 dilution, T5168) was from Sigma Aldrich (St.Lois, MO, US). Alexa 488 goat anti-mouse, Alexa 546 goat anti-mouse, and Alexa 488 goat anti-rabbit were from Invitrogen (Carlsbad, CA, USA). Hoechst and Alexa-488 Phalloidin was from Molecular probes (Invitrogen, Paisley, UK). Western blot: Mouse anti-Grp75 (Hsp70) (ab13529, 1:2000 dilution), rabbit anti-PARK7 (ab37180, 1:2000 dilution), rabbit anti-actin (1:500 dilution, ab1801), rabbit anti-desmin (1:2000 dilution, ab8592) were from Abcam. Mouse anti-α-tubulin (1:10 000 dilution, T5168) was from Sigma Aldrich (St. Lois, MO, US). CY3-conjugated goat anti-mouse and CY5-conjugated goat anti-rabbit (1:2500 dilution, PA43010 and PA45011, respectively), were procured from GE Healthcare (Buckinghamshire, UK). JC-1 Mitochondrial membrane potential probe was from Thermo Fisher Scientific (Waltham, MA, US) and Z-LEHD-FMK (irreversible caspase-9 inhibitor, 20 μM) was from Abcam (Cambridge, UK)).