Plin4

For SDS-PAGE followed by immunoblotting, protein was extracted from 25 mg cerebellar tissue of 12-week-old mice. The tissue was homogenized with a motor pestle in 10 vol. RIPA buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Igepal CA-630 (Sigma), 0.5 % sodium deoxycholate, 0.1 % SDS, 1 mM PMSF, Complete Protease Inhibitor Cocktail (Roche)] and incubated on ice for 15 min. After centrifugation at 4 °C and 16,000×g for 20 min, the supernatant was stored (RIPA-soluble fraction), and the remaining pellet was dissolved in ½ vol. 2 × SDS buffer [137 mM Tris–HCl (pH 6.8), 4 % SDS, 20 % glycerol, Complete Protease Inhibitor Cocktail (Roche)] by sonification followed by 10 min of centrifugation at 16,000×g. The resulting supernatant was stored as RIPA-insoluble fraction. Protein concentration was determined with the BCA protein assay kit (Interchim, France), and 20 μg of each sample were loaded onto a 7.5 % polyacrylamide gel. After gel electrophoresis, the proteins were transferred to a PVDF membrane by wet blotting. The membranes were blocked with 5 % slim milk powder in PBST and incubated with antibodies against PLIN4 (1:500, Novus Biologicals), WDFY1 (1:500, Life Span BioSciences), CNTN4 (1:1,000, Abcam), or β-ACTIN (1:10,000, Sigma). ECL (Pierce) was used for visualizing the bands, which were subsequently quantified via densitometric analysis with ImageJ.

Plin4 (Novus, Cat 13776,1:500 for IF &1:1000 for WB); TH (Abcam, Cat ab6211,1:800 for IF); GFAP (Millipore, Cat MAB360,1:500 for IF); poly-Ub (CST, Cat 3936,1:500 for IF & 1:1000 for WB); Tom20 (CST, Cat 13929s,1:500 for IF & 1:1000 for WB);LC3B (CST, Cat 2775,1:500 for IF & 1:1000 for WB); p62 (CST, Cat 5114,1:500 for IF & 1:1000 for WB); Parkin(Abcam, Cat ab15954,1:800 for WB), Cytochrome C (CST, Cat 4272s,1:800 for WB), AIF(E-1) (Santa Cruz, Cat sc-13116,1:800 for WB), caspase-3 (CST, Cat 9662s,1:500 for WB), Beclin-1(CST, Cat 3738,1:1000 for WB), ATG7 (ABGENT, Cat AP1813a,1:800 for WB), β-actin (Santa Cruz, Cat SC-47778,1:1000 for WB). Secondary antibodies: Alexa F488 Donkey anti-mouse (Invitrogen, Cat A21202, 1:1000), Alexa Fluor 488 Goat anti-Rabbit (Invitrogen, Cat A11008,1:1000), Alexa Fluor 594 Donkey anti-Goat (Invitrogen, Cat A11058, A11008, A 11059,1:1000), HRP-conjugated secondary antibodies (Thermo, Cat 0031430, 31460, 31402, 1:1000).