Revert total protein stain kit

For mitochondrial OXPHOS protein expression, proteins were extracted in RIPA buffer, and protein concentration was determined with the BCA assay (Pierce). Cell lysates were then diluted in SDS-PAGE loading buffer (Roti-Load, K929.1, Carl Roth), and SDS-PAGE was performed on 20 μg of cell lysate per sample on Tris-glycine-SDS polyacrylamide gels (4561095, Biorad), then transferred to PVDF (1620177, Biorad) using standard techniques. Primary antibodies used were against the mitochondrial respiratory chain subunits (mitochondrial OXPHOS antibody cocktail containing cytochrome c oxidase subunit 2 [COX2], cytochrome b-c1 complex subunit 2 [UQCRC2], succinate dehydrogenase [ubiquinone] flavoprotein subunit B [SDHB], NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 [NDUFB8] and ATP synthase subunit alpha [ATP5A]; Cat. #ab110411, Abcam). Total protein detection (REVERT Total Protein Stain Kit, P/N 926-11010; Li-Cor) was used to normalize total cellular protein. Primary antibodies were detected with appropriate anti-mouse or anti-rabbit Infared IRDye 680 RD or 800 CW antibodies (Abcam). Protein band intensities were detected with the Odyssey 3.0 (Li-Cor Biosciences) and quantified using Image Lab 6.0 software (Biorad), and band intensity determined in the linear range was normalized to the band intensity of total protein.

Ten micrograms of exosomal proteins were loaded onto a commercial 4–20% SDS/PAG gradient gel. The electrophoresis was run at 100 V for 75–80 min until the dye-front migrated to 0.5 cm from the bottom of the gel. Resolved proteins on the gel were transferred onto a PVDF membrane by electro transfer unit at 60 V for 90 min. The membrane was stained/destained for total protein staining according to the manufacturer’s protocol (REVERT Total Protein Stain kit, LI-COR; Cat# 926-11016). The membrane was blocked with a blocking agent (SEA BLOCK Blocking, Thermo Scientific, Cat # UH2788881) for 1 h at room temperature, followed by overnight incubation with anti- TDP-43 Ab (1:1000 dilution; Proteintech Cat# 10782-2-AP), anti-P(S409/410) TDP-43 Ab (1:1000 dilution; Proteintech Cat# 66318-1-Ig), and anti-SOD1 Ab (1:750 dilution; Proteintech Cat#10269-1-AP) on an orbital shaker at 4 °C. The next day the membrane was incubated with Infrared (IR)-tagged antibody (1:10,000 dilution; LI-COR Cat# 926-3211, 926-68071, 926-32210, 926-68070) for 1–2 h at room temperature. The protein bands were visualized in an image analyzer (LI-COR Odyssey Infrared Imager (Model No. 9120). The band intensity was normalized based on total protein staining signals, and analyzed by Image Studio image analyzing program (V.3.1, Li-COR Biosciences).

Heads of 5 and 55 day-old females (three biorepeats of 20 heads per timepoint for each age) were homogenized in Laemmli buffer, sonicated, boiled at 100 °C for 5 min and centrifuged at 12,000g at 4 °C. A constant ratio of the buffer (7 μl per head) was used to ensure equal protein loading and separation on NuPAGE 4–12% gradient acrylamide gel (Life Technologies). Proteins were transferred to the 0.45 μm polyvinylidene fluoride (PVDF) Immobilon-FL membrane (Millipore Billerica, MA) and stained for 5 min with REVERT Total Protein Stain Kit (Li-Cor Biosciences). After staining, membranes were scanned in the 800 channel on the Odyssey Infrared Imaging system to quantify the total protein. The staining was reversed using the same kit, and the membranes were incubated in 1 × TBST (10 mM Tris, 0.15 M NaCL, 0.1% Tween-20, pH 7.5)+5% dry milk for 2 h, then overnight at 4 °C with 1:15,000 anti-PER (gift from Dr J. Price)39 (link) in blocking buffer. Membranes were treated for 2 h with 1:20,000 goat anti-rabbit IRDye800 (catalogue # 926–32211, LI-COR Biosciences, Lincoln, NE) diluted in Odyssey Blocking Buffer. After washes, PER signal was quantified relative to total protein using the LI-COR Odyssey Infrared Image Studio software according to the manufacturer's instructions. Uncropped version of the PER western blot is present in Supplementary Fig. 3.