Pegfp c2

To express GFP-fused chicken CENP-A, the cDNA encoding full-length chicken CENP-A was cloned into pEGFP-C2 (Clontech) (ggCENP-A_pEGFP-C2). The cDNA fragment of the chicken CENP-A^QD mutant, which has two amino acid substitutions (L67Q and L68D), was synthesized by Gene Synthesis (FASMAC), and was cloned into pEGFP-C2 to yield the GFP-CENP-A^QD expression vector (ggCENP-A^QD_pEGFP-C2).

GFP-tagged constructs, including Cdc42-WT, Cdc42V12, dominant negative (DN) Cdc42N17 and RhoAV14, were cloned into pEGFP-C2 (Takara Bio Inc., Shiga, Japan). WT-PAK1 and DN PAK1 (H83/86L, K299R) cDNAs were cloned into pCMV-myc (Takara Bio Inc.). DH domain constructs were cloned into pEGFP-C2 using polymerase chain reaction (PCR). The inserts corresponded to amino acids 100–276 of wild-type (DH^WT) or L238R/L239R (DH^mt) βPIX [18] (link) or to amino acids 1048–1239 of wild-type (DH^WT) or L1048R/L1049S (DH^mt) Tiam1.

According to the manufacturer’s instructions, to construct plasmids expressing the viral protein, DHAV-1 RNA was isolated using RNAiso Plus Reagent (TaKaRa). According to the manufacturer’s instructions, genomic DNA was then removed, and reverse transcription was performed using a PrimeScriptTM RT Reagent Kit (TaKaRa). VP0, VP1, 2A, 2B, 2C, 3AB, and 3D sequences were amplified from DHAV-1 cDNA with PCR and primers (Table 1). VP0, VP1, 2A, 2B, 2C, and 3D were integrated into the pCAGGs vector with a one-step cloning kit (Vazyme). 3AB was cloned into the pCMV-Myc vector with the DNA Ligation Kit (TaKaRa). The eukaryotic expression vector pCAGGs was gifted by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The eukaryotic expression vector pCMV-Myc was purchased from TaKaRa. pCAGGs-VP3-Flag and pEGFP-N1-3C were also stocks in our laboratory (Lai et al., 2019 (link); Sun et al., 2019 (link)). Duck-derived G3BP1 gene was cloned into the pEGFP-C2 vector with the DNA Ligation Kit (TaKaRa). The pEGFP-C2 vector was purchased from YouBio.

Macaque fecal samples were collected at a zoological park in Yunnan Province, China. Adenoviruses were isolated from these as described below. HEK 293 cells were purchased from the American Type Culture Collection (CBP60434). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal calf serum (Gibco) at 37°C, in 5% CO₂. Escherichia coli DH5α and E. coli BJ5183 competent cells were purchased from Stratagene (La Jolla CA, USA). Plasmid PBR322 and pEGFP C2 were purchased from TAKARA Co. (Dalian, China). Q5 DNA polymerase, T5 exonuclease, Phusion DNA polymerase, Taq DNA ligase, and restriction enzymes were from New England Biolabs (Beijing, China) and used according to the manufacturer’s instructions. Fluorescent quantitative PCR kits for HAdVs were purchased from Guangzhou Huayin Corp. (Guangzhou, China). The 5× isothermal reaction buffer used in the Gibson assembly method (48 (link), 49 (link)) contained the following: 3 mL of 1 M Tris-HCl (pH 7.5), 150 µL of 2 M MgCl₂, 600 µL of 10 mM dNTP, 300 µL of 1 M DTT, 1.5 g PEG-8000, and 300 mL of 100 mM NAD. Subsequent Gibson assembly master cocktails contained 40 µL 5× isothermal reaction buffer, 0.2 µL of 10 U µL⁻¹ T5 exonuclease, 2.5 µL of 2 U µL⁻¹ Phusion DNA polymerase, 20 µL of 40 U µL⁻¹ Taq DNA ligase, and distilled water, to a final volume of 150 µL. All reagents were stored at −20°C.

To identify the characteristics of the interaction of XIAP mutants with RIP2 in live HEK 293 T cells, we prepared an N-terminally green fluorescent protein (GFP)-tagged XIAP-WT construct (pEGFP–XIAP-WT). The cDNA clones containing full-length human RIP2 (KUGI #hMU008516) and XIAP (KUGI #hMU005178) coding sequences were purchased from the Korea Human Gene Bank (KHGB). The PCR-amplified full-length XIAP open reading frame (ORF) was digested with SalI and BamHI, and subcloned into the XhoI and BamHI sites of pEGFP-C2 (Takara Bio, USA). For site-directed XIAP-C203Y, -ΔG204, -R166I, -R166K, -W173G, -G188E, -L189P, -V198M, -L207P, and -H220Y mutagenesis, the whole plasmid from pEGFP–XIAP-WT was PCR-amplified, digested with DpnI to remove the template, and then cloned by self-ligation. To produce the N-terminal red fluorescent protein (RFP)-tagged RIPK2-WT-expressing construct (pmRFP–RIP2-WT), pmRFP-C2, an mRFP expression vector, was generated using the pMRS reporter construct (ToolGen, Seoul, Korea), and the PCR-amplified full-length RIP2 ORF was subcloned into the BglII and EcoRI sites of pmRFP-C2.