artificially synthesized gene sequence  (GenScript corporation)

Journal: Oncotarget

Article Title: Newcastle disease virus employs macropinocytosis and Rab5a-dependent intracellular trafficking to infect DF-1 cells

doi: 10.18632/oncotarget.13345

Figure Lengend Snippet: ( A – B ) DF1 cells were treated with NH 4 Cl or Baf A1 for 4 h before or after NDV infection, and cells lysates were prepared for western blot analysis. The NP/b-actin ratio is shown. ( C ) Intracellular trafficking of NDV requires Rab5a. DF-1 cells were transfected with pEGFP, pEGFP-Rab5wt or pEGFP-Rab5S34N, infected with 5 MOI NDV for 4 h at 37°C and analyzed by western blotting. The NP/b-actin ratio is shown. * P < 0.05. ( D ) Rab7a function is dispensable for NDV intracellular trafficking. DF-1 cells were transfected with pEGFP, pEGFP-Rab7wt or pEGFP-Rab7T22N, and analyzed using Western blotting as in C. ( E ) NP co-localized with early endosome marker EEA-1-positive vesicles. DF-1 cells were infected with 5 MOI NDV for 1 h and observed using CLSM: EEA1 (red), NP (green), and cell nucleus (blue). Scale bar = 20 μm.

Article Snippet: The open reading frame of wild-type (wt) Rab5a (Genbank accession number: NM_001006363.2), Rab7a (Genbank accession number: XM_003641978.3), and dominant-negative (DN) GTP-binding defective Rab5a (S34N) and Rab7a (T22N) gene sequences were artificially synthesized (Sangon Biotech, Shanghai, China) and separately subcloned into a pEGFP-C3 plasmid vector (Takara Biomedical Technology Co., Ltd. Dalian, China).

Techniques: Infection, Western Blot, Transfection, Marker