Pa45011

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)).

Supernatant S1 fractions (10 μg protein) from synaptosome purifications were used for Western blotting (control n = 5, methamphetamine self-administration n = 5). All of these samples were represented in the LC-MS/MS analysis and were chosen based on sufficient protein amount for Western blot analyses. One sample from each group had insufficient protein amount and therefore were excluded. Electrophoresed proteins were transferred to an Immobilon-FL PVDF membrane (Millipore, Thermo Fisher Scientific), blocked with 5% BSA and probed using anti-phosphatidylethanolamine binding protein-1 (Pebp1, 1/750 dilution, ab76582, Abcam) and anti-amphiphysin (Amph, 1/25,000 dilution, ab52646, Abcam) followed by anti-rabbit Cy5 (PA45011, GE Healthcare) and imaged using a Fujifilm FLA-5000 fluorescent scanner. Membranes were re-probed and normalised to α-tubulin (ab18251, Abcam). One-tailed t-tests were used to identify significant changes between control and methamphetamine groups (p<0.05).