Pcmv 3ha

Human CCL2 and bovine CCL2 cDNAs were cloned into the vector pCMV-3HA (Clontech, Mountain View, CA, USA). The mature human CCL2 was cloned into pCMV-3HA after removing the N-terminal 23 amino acids of the human CCL2 precursor.
The BFV infectious clone (pBS-BFV-Z1), pCMV-3HA-BEnv, pCE-puro-3Flag-BGag, and pCE-puro-3Flag-Lck-BGag were previously described [12 (link),19 (link)]. Mutations were generated by site-directed polymerase chain reaction (PCR) (Toyobo, Osaka, Japan). All mutant plasmids were verified by DNA sequencing (Genewiz, Beijing, China) before use.
Antibodies used for protein analysis were as follows: anti-Flag, anti-HA, anti-Tubulin, and secondary antibodies labeled with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). With reference to the established laboratory protocol for preparing antibodies [37 (link)], antibodies against human CCL2 were generated in BALB/c mice using bacterially purified human CCL2 (24–99aa) protein as immunogens. Because human CCL2 (1–99 aa) forms CCL2 (24–99 aa) after being cleaved by signal protein, CCL2 antibody can be used to detect two forms of CCL2.

Stable MKN28 and AGS cell lines with doxycycline-dependent tetracycline-inducible (tet-on) KIAA1324 WT, KIAA1324 3NQ, and KIAA1324 ΔTM were established through a retroviral system. The human KIAA1324 gene was cloned into the vector pCMV-3HA (Clontech), and the virus was produced by inserting 3HA-tagged KIAA1324 DNA into a retroviral vector, pRetroX (Clontech). MKN28 and AGS cells were infected with this virus and underwent 2 μg/mL G418 and puromycin selection.

Human ALIX and human TSG101 cDNAs were cloned into vector pCMV-3HA (Clontech, Mountain View, CA, USA). Different site mutants (pCMV-3HA-Alix V498D, pCMV-3HA-Tsg101 Y63A, and N69P) were generated using site-directed mutagenesis (Toyobo, Osaka, Japan) according to the manufacturer’s recommendations.
The primers sequence used to construct the pCMV-3HA-Alix V498D mutant were: forward primer—5′-GGAACCAACTTCAGAACAGATTTAGATAAAGCTGTGCAG-3; reverse primer—5′-CTGCACAGCTTTATCTAAATCTGTTCTGAAGTTGGTTCC-3′. The primers sequence used to construct the pCMV-3HA-Tsg101 Y63A mutant were: forward primer—5′-GGAACAATCCCTGTGCCTGCTAGAGGTAATACATAC-3; reverse primer—5′-GTATGTATTACCTCTAGCAGGCACAGGGATTGTTCC-3′. The primers sequence used to construct the pCMV-3HA-Tsg101 N69P mutant were: forward primer—5′-GAGGTAATACATACCCTATTCCAATATGCCTATGG-3; reverse primer—5′-CCATAGGCATATTGGAATAGGGTATGTATTACCTC-3′.
The coding sequence of BFV Env was inserted into pCMV-3HA to construct the pCMV-3HA-BEnv plasmid, and the coding sequence of BFV Gag was cloned into pCE-puro-3×FLAG to construct the pCE-puro-3×FLAG-BGag plasmid. Mutations were generated by designing specific mutation primers and using site-directed polymerase chain reaction (PCR) (Toyobo, Osaka, Japan).
All mutant plasmids were verified by sequencing before use (Genewiz, Beijing, China).