Protein lysates were processed for immunoblots as described [24 (link)]. Primary antibodies include: CHOP (Santa Cruz sc-7351 for flow cytometry or Proteintech 15204–1-AP for western blot), PARP (Cell Signaling Technology 9542), and Calnexin (loading control; Enzo ADI-SPA-865). Nanoluciferase activity was assessed using the Nano-Glo In-Gel Detection kit (Promega, USA) following the manufacturer’s protocol. qRT-PCR, including primer validation by standard curve and melt curve analysis, was also as described [24 (link)]. Briefly, RNA was isolated using Trizol (Thermofisher, USA) following the manufacturer’s protocol, and RNA concentrations were evaluated using the Qubit RNA Broad Range kit (Invitrogen, USA). 400 ng of RNA was used for reverse transcription using PrimeScript RT Master Mix (Takara, USA). PCR reactions were performed using TB Green Premix Ex Taq (Takara, USA). Gene expression was normalized against the average of two loading controls (Btf3 and Ppia).
qRT-PCR Primers:
Btf3 forward: CCAGTTACAAGAAAGGCTGCT reverse: CTTCAACAGCTTGTCCGCT
Ppia forward: AGCACTGGAGAGAAAGGATT reverse: ATTATGGCGTGTAAAGTCACCA
FLuL exon1–2 forward: TTGAAGATGAGCGGGTGGCA reverse: CTTTCAGGTGTGGTGGTGTA
FLuL exon 2-nLuc forward: GTGTTCCAGAAGGAAGTGCA reverse: CTTTGGATCGGAGTTACGGA
FLuL exon 3–4 forward: GGAAGCCTGGTATGAGGAT reverse: CCACTCTGTTTCCGTTTCCT