RNA extraction and real-time quantitative PCR

For quantitative PCR (qPCR), total RNA was isolated from fly heads, cell cultures, or mouse brain tissues using TRIzol (Invitrogen) according to the manufacturer’s instruction. After DNase (Promega) treatment to remove genomic DNA, the RT reactions were performed using All-in-One cDNA Synthesis SuperMix kit (Bimake). The cDNA was then used either for semiquantitative RT-PCR experiments by PCR amplification using the Taq Plus Master Mix (Vazyme) or real-time qPCR using the SYBR Green qPCR Master Mix (Bimake) with the QuantStudio™ 6 Flex Real-Time PCR system (Life Technologies). The mRNA levels of actin or GAPDH were used as an internal control to normalize the mRNA levels of genes of interest. The qPCR primers used in this study are listed below:

hβ-actin forward: 5′-GTTACAGGAAGTCCCTTGCCATCC-3′

hβ-actin reverse: 5′-CACCTCCCCTGTGTGGACTTGGG-3′

hPINK1 forward: 5′-CCCAAGCAACTAGCCCCTC-3′

hPINK1 reverse: 5′-GGCAGCACATCAGGGTAGTC-3′

hParkin forward: 5′-GTGTTTGTCAGGTTCAACTCCA-3′

hParkin reverse: 5′-GAAAATCACACGCAACTGGTC-3′

hTDP-43 forward: 5′-GGGAAATCTGGTGTATGTTGTCA-3′

hTDP-43 reverse: 5′-GAAAATCACACGCAACTGGTC-3′

PINK1-V5 forward: 5′-CAGACGTGAGACAGTTGGTG-3′

PINK1-V5 reverse: 5′-GTAGAATCGAGACCGAGGAGAG-3′

Flag-Parkin forward: 5′-CAAGGATGACGACGATAAGT-3′

Flag-Parkin reverse: 5′-GCTGGAAGATGCTGGTGT-3′

mGAPDH forward: 5′-CACCATCTTCCAGGAGCGAG-3′

mGAPDH reverse: 5′-CCTTCTCCATGGTGGTGAAGAC-3′

mPINK1 forward: 5′-CACACTGTTCCTCGTTATGAAGA-3′

mPINK1 reverse: 5′-CTTGAGATCCCGATGGGCAAT-3′

mParkin forward: 5′-TCTTCCAGTGTAACCACCGTC-3′

mParkin reverse: 5′-TCTTCCAGTGTAACCACCGTC-3′

dActin forward: 5′-GAGCGCGGTTACTCTTTCAC-3′

dActin reverse: 5′-GCCATCTCCTGCTCAAAGTC-3′

dPINK1 forward: 5′-GAGCAACAGCAGCATCAGAA-3′

dPINK1 reverse: 5′-TGATGTTTGAATTCGCTGGA-3′

dParkin forward: 5′-AGCGATGCCACGACAATAGAGC-3′

dParkin reverse: 5′-GCGAAGGTTCCTCCTCCTCCAA-3′