Pcmv6 entry

HeLa cells were cultured in a growth medium (Dulbecco's modified Eagle's medium containing 10% fetal calf serum) at 37 °C in a 5% CO₂ atmosphere. pCMV6‐TMEM160‐Myc‐DYKDDDDK (OriGene, Rockville, MD, USA), which expresses the TMEM160‐Myc‐DYKDDDDK protein with a DYKDDDDK‐tag at the C‐terminus, was transfected into HeLa cells using Lipofectamine 3000 (Invitrogen) and Opti‐MEM I (Gibco, Grand Island, NY, USA). Control cells were obtained by transfecting HeLa cells with pCMV6‐Entry (OriGene), an empty vector. After 72 h of culture, the cells were harvested and suspended in a growth medium containing 1 mg·mL⁻¹ G418. The cell suspension was diluted stepwise in a 96‐well plate. Each single colony resistant to G418 was selected and further cultured as a stable transformant. The cells were harvested in ice‐cold phosphate‐buffered saline (PBS), containing 1 mm ethylenediaminetetraacetic acid, and washed twice with PBS. The cells were lysed with PBS containing 1% Triton X‐100 and centrifuged at 10 000 × g for 5 min. The supernatant was mixed with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS/PAGE) loading buffer for immunoblotting analysis to evaluate the expression of TMEM160‐Myc‐DYKDDDDK.

HEK293 cells (European Collection of Authenticated Cell Cultures) were grown in high glucose Dulbecco’s modified Eagle medium (Life Technologies) supplemented with 10% heat inactivated foetal bovine serum (FBS; Sigma), penicillin/streptavidin (Invitrogen) and l-glutamine (Invitrogen). HEK293 cells were transfected with RNAiMAX (Invitrogen) and 2.5 µM Silencer® Select Validated UBA1 siRNA (s601, targeted against exons 24 and 25; Life Technologies), or with Lipofectamine™ (Invitrogen) and 3.5 µg pCMV6-XL4-UBA1 plasmid (Origene) for proteomics samples. For proteomics, three separate transfections were combined per sample with three samples per condition. For co-transfection of multiple plasmids, 1.25 µg of each plasmid was used and the same amount of DNA and Lipofectamine™/RNAiMAX was added to each well: pCMV6-XL4-UBA1 (Origene), pCMV6-Entry (Origene); pEGFP-GARS-N2 (gift from Dr Antonelli Antonellis), pEGFP-N1 (gift from Prof. Mike Cousin); pcDNA3.1-HA-Ubiquitin (gift from Prof. Yeh), Addgene plasmid #18712 (Kamitani et al., 1997 (link)), pcDNA3.1+ (gift from Prof. Mike Cousin); pcDNA3.1-SMN-V5-His. Negative control 2 siRNA (Life Technologies) was used as a control for UBA1 siRNA. For all co-transfections, cells were harvested 48 h after transfection.

The full-length cDNA of AKAP3, TSSK4, ODF2, and QRICH2 was synthesized and separately cloned into pENTER (Vigene), pCMV6-Entry (Origene), pCMV6-Entry, and pcDNA^TM3.1⁽⁺⁾ (Invitrogen) vectors containing a FLAG, Myc, and HA tag sequence. CABYR and ODF2 promoter fragments were amplified and cloned into the pGL3-Basic luciferase reporter vector (Promega). The shRNAs designed to interfere with QRICH2 expression and the negative control SuperSilencing shRNA (shNC) were synthesized and cloned into the psi-LVRU6GP vector by GeneCopoeia. The target sequences of QRICH2 shRNAs were 5′-GGGTTCACTTCCTTAACATCA-3′. The primers for promoter region amplification are listed in Supplementary Table 5.

Rat P2X2 (rP2X2) (Brake et al., 1994 (link)) cDNA in pcDNA1 was generously provided by Dr. David Julius (the University of California, San Francisco, CA). Mouse P2X5 (mP2X5) cDNA in pCMV6-Entry was purchased from OriGene and subcloned into pcDNA3.1. A previously characterized rP2X2 construct where three native cysteines (C9, C348, and C430) were mutated to threonine (rP2X2–3 T) was used as a background construct for MTS experiments because it is insensitive to MTS reagents yet displays functional properties that are similar to the wild-type channel (Li et al., 2008 (link)). All mutations in rP2X2 and mP2X5 were made using the QuikChange Lightning technique (Agilent Technologies) and confirmed by DNA sequencing (Macrogen).