Real-Time Quantitative PCR (RT-qPCR) and Western Blot

To examine the relative expression levels of PtCGI-58 mRNA in WT and transformants, cells cultured for 4, 6 and 8 days were harvested by centrifugation at 3,000 ×g for 10 min and used for RNA extraction with Trizol reagent (TaKaRa, China). The first-strand cDNA reverse transcribed from RNA according to the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, R312-01/02, China) was used as template for RT-qPCR using LightCycler 480 SYBR Green I Master (Roche, Germany) and a LightCycler 480 Real-Time PCR System (Roche). The primers used for RT-qPCR were shown in the Supplemental Table. The relative expression quantification of the target gene was calculated using histone H4 as the endogenous control gene [25] according to the 2^−ΔΔCt method [26].

Cells grown for 3 days were harvested by centrifugation for protein extraction using Western/IP lysis buffer (Beyotime, China), and the protein concentration was determined with the BCA Protein Concentration Assay Kit (Beyotime). The protein samples were isolated on a 12% SDS-PAGE gel before being transferred to PVDF membrane and hybridized with PtCGI-58 polyclonal antibody prior to HRP-labeled goat anti-rabbit IgG secondary antibody. After extensive washing, the blots were developed by ECL (Millipore, USA), and chemiluminescence was captured on an ImageQuant LAS 4000 mini apparatus (GE Healthcare Life Sciences, UK).