Ythdf3 25537 1 ap

Fibroblasts from the TUH sporadic PD cohort and healthy individuals were plated on 10 cm diameter dishes at the same time at a density of 300,000 cells per dish, and grown in DMEM for two days following which they were treated for 16 h with 100 nM MLi-2 or DMSO as indicated. Cells with a passage number of 10-12 were lysed in Laemmli SDS PAGE buffer (ThermoFisher Scientific) and loaded onto a SDS-PAGE gel alongside a normalisation standard which consisted of 5 µg of fibroblast lysate. Proteins were transferred to nitrocellulose membranes using wet transfer as previously described^{12 (link)} and incubated at 4 °C overnight with the following antibodies and dilutions; AP2B1 #15690-1-AP (1:4000), YTHDF3 #25537-1-AP (1:3000), ATG9A #67096-1-Ig (1:5000) all from ProteinTech Group Inc. (Rosemont, USA) and EHD1 #MA5-42814 (1:1000), (ThermoFisher Global) and ABHD5 (1:100) (#SC-376931; Santa Cruz Biotechnology, Dallas, USA). Secondary antibodies LiCor IRDye 800Cw goat anti-rabbit and Li-Cor IRDye 680RD donkey anti-mouse (LI-COR Biosciences, Lincoln, USA) were used at a dilution of 1:15,000 in 5% milk in Tris buffered saline (Tris (20 mM), NaCl (150 mM), Tween-20 (0.1% w/v)) and were incubated with the membranes for 1 h followed by washing. Digital images of fluorescence intensities were acquired with the LI-COR Odyssey imaging device.

Cells or tissues were homogenized in an l-RIPA lysis buffer (R0020, Solarbio). Protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblotted with the indicated antibodies, and visualized using the ECL. The antibody information and dilutions were as follows: METTL3(D2160,Cell Signaling Technology),1:3000; Akt(9272, Cell Signaling Technology),1:5000; p-Akt (S473)(9271, Cell Signaling Technology),1:5000; UCP1(U6382, Sigma),1:5000; PGC-1a(66369-1-lg, Proteintech),1:1000; β-Actin(60008-1-lg, Proteintech),1:5000; PPARg(16643-1-AP, Proteintech),1:300 0; GAPDH(60004-1-lg, Proteintech),1:5000; Total OXPHOS (ab110413, Abcom),1:2000; PRDM16(A11581, ABclonal),1:1000; YTHDF1(17479-1-AP, proteintech),1:2000; YTHDF2(24744-1-AP, proteintech),1:5000; YTHDF3(25537-1-AP, proteintech),1:3000.