Fast mutagenesis system kit

Human embryonic kidney 293 (HEK 293) cells were cultured in DMEM supplemented with 10% FBS and 0.5% PS in 5% CO₂ at 37°C. Expression vectors were constructed by cloning human LMNA into pEGFP-N1. Utilizing standard PCR-based in vitro mutagenesis (Fast Mutagenesis System Kit; TransGen Biotech, Beijing, China), wild-type LMNA was altered to express four mutants: c.143G>C (p.R48P), c.745C>T (p.R249W), c.1117A>G (p.I373V), and c.IVS8-7_14del (p.I497_E536del). Each mutant full-length construct was confirmed by direct DNA sequencing. HEK 293 cells were transfected using Lipofectamine Plus (Invitrogen, Carlsbad, California, USA). Transfected cells were grown at 37°C, harvested at 24 h after transfection, and observed under microscopy. Expression of green fluorescent protein (GFP) in cells containing the wild-type and mutant proteins was observed to determine lamin A/C localization.

All the site-directed or C terminus-truncated variants of UPF0118 were constructed using pET22b-P-UPF0118 as a template and the corresponding primers listed in Supplementary Table S1 via a Fast Mutagenesis System kit purchased from TransGen Biotech Co., Ltd. (Beijing, China), as described in our recent studies (Shao et al., 2018 (link); Xu et al., 2019 (link)). All the final UPF0118 variants were re-sequenced to confirm the accuracy of mutagenesis, and the corresponding plasmids were transformed into E. coli KNabc for growth tests and Na⁺(Li⁺)/H⁺ antiport activity assays.

Recombinant protein purification was performed as described previously^{83 (link)}. Wild‐type FABP5 was cloned into pGEX6P1 and expressed as glutathione‐S‐transferase (GST) fusion proteins with a TEV protease cleavage site in between. FABP5 C127S was generated with a Fast Mutagenesis System kit (Transgen, FM111) based on pGEX6P1‐FABP5 WT. GST fusion proteins were expressed in E. coli BL21 (DE3) at 16 °C to achieve maximal soluble expression. Cells were collected by centrifugation and washed three times with cold PBS. The cells were lysed by sonication in lysis buffer (20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.5% Triton‐X100, protease inhibitor cocktail from Roche) and centrifuged at 12,000 g for 15 min. GST‐Sepharose resin (0.2 mL; GE Healthcare) pre‐equilibrated with 20 mL TEV protease cleavage buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% NP‐40, 1 mM DTT) was added to the supernatant and rotated at 4 °C for 2 h. Next, beads were washed three times with TEV protease cleavage buffer, and then the recombinant protein was eluted from the resin by incubation overnight at 4 °C with 10 μg/mL TEV protease to cleave off the desired protein from the GST tag, which was still bound to the GST‐Sepharose resin after the overnight cleavage reaction.

Expression vector pcDNA3.1(+) was purchased from Invitrogen (Carlsbad, CA, USA). A fast mutagenesis system kit was purchased from Transgen Biotech (Beijing, China). An Exfect 2000 transfection reagent was obtained from Vazyme Biotech (Nanjing, China). DMEM/F-12 1:1 medium was purchased from Thermo Fisher Scientific (Beverly, MA, USA). Reporter gene plasmids pGL4.29[luc2P/CRE/Hygro] and pGMLR-TK were purchased from Promega (Beijing, China). A dual-luciferase reporter gene assay kit was purchased from Beyotime Biotechnology (Shanghai, China). PMSF and protease inhibitors were purchased from Solarbio Life Science (Beijing, China). c-myc Rabbit mAb was obtained from Abcam (Cambridge, UK). Goat anti-rabbit IgG-HRP was purchased from Cell Signaling Technology (Boston, MA, USA).

To construct plasmids of pCB-TMV-fsCP, pCB-TMV-CGfsCP and pCB-TMV-PMfsCP (Supplementary Figure S1A) that cannot express the respective viral coat proteins, pCB-TMV-SY, pCB-CGMMV-LN and pCB-PMMoV-HLD were used as templates and were amplified by primer pairs TMVfsCP + /TMVfsCP-, T-CGCP-fsCP + /T-CGCP- fsCP-, T-PMCP-fsCP + /T-PMCP- fsCP-, respectively. The amplified DNA fragments were treated with DMT enzymes and subjected to self-ligation using a Fast Mutagenesis System kit (TransGen, Biotech, China). The constructs were transformed into Agrobacterium tumefaciens GV3101. All of the PCR primers used in this study are listed in Supplementary Table S1.