The specimens were viewed using a Tecnai G2 transmission

The specimens were viewed using a Tecnai G2 transmission

electron microscopy at 75 keV. For Western blot, 10 μg purified VLPs were separated by SDS-PAGE electrophoresis and subjected to Western blot assay. (F, H) Electron micrograph images and Western blotting result of VLP H2. (I) RGD-core-IFN-α2a fusion protein bind with breast cancer cells MDA-MB231. Then, 0.2, 0.5, 2, 5, and 10 μM fusion proteins His-H1, His-H2, His-H3, and His-H4 were co-incubated with MDA-MB231 at 37° under 5% CO2. After 2 h, the cells were washed three times with PBS, and green fluorescence was observed under the fluorescence microscope. Scale bar = 100 μm. Binding specificity assay MDA-MB231 human breast cancer cells were cultured Selleck MK-4827 in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin, at 37°C under 5% CO2. Then, 0.2, 0.5, 2, 5, and 10 μM fusion proteins His-H1, His-H2, His-H3, and His-H4 were co-incubated with MDA-MB231 at 37°C under 5% CO2. After 2 h, the cells were washed click here three times with PBS, and green fluorescence was observed

under the fluorescence microscope. Construction of recombinant baculovirus pH1, pH2, pH3, and pH4 were digested by BamHI/EcoRI and were subcloned into pFastBac dual vector (pFBD) that had been pre-treated with BamHI/EcoRI and produced pFBD-H1, pFBD-H2, pFBD-H3, and pFBD-H4. The four donor plasmids (pFBD-H1, pFBD-H2, pFBD-H3, and pFBD-H4) mediated the insertion of genes into the AcBacmid by Tn7-mediated transposition to generate AcFBD-H1, AcFBD-H2, AcFBD-H3, and AcFBD-H4 bacmids, respectively. These recombinant bacmids were confirmed by PCR and then were introduced by transfection into Sf9 cells to produce the recombinant viruses vAcH1, vAcH2, vAcH3, and vAcH4. Real-time Q-PCR and Western blotting Total

RNA was extracted from cells with PureLink RNA kit (Life Technologies Corporation). cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen, Carlsbad, CA, USA) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Thalidomide Biosystems, Foster City, CA, USA). The relative quantification of gene expression for each sample was analyzed by the ΔC t method. The following primers were used to amplify HCV core: 5’- GCC CAC AGG ACG TCA AGT −3’ and 5’- CGC AAC CCT CAT TGC CAT −3’; 18S rRNA: 5’-ACC TGG TTG ATC CTG CCA GT-3’ and 5’-CTG ACC GGG TTG GTT TTG AT-3’. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated on a 12% sodium SB525334 mouse dodecyl sulfate polyacrylamide gel (SDS-PAGE) by electrophoresis and subjected to Western blot assay.

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