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Figure 2 | Virology Journal

Figure 2

From: Lack of Processing of the Expressed ORF1 Gene Product of Hepatitis E Virus

Figure 2

Expression of US swine HEV ORF1 and rubella virus non-structural ORF. 293T cells were transfected with 5 μg of pCMV-SHEV DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 12, 24, and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. The cells were washed twice with PBS and lysed with 1X NP-40 buffer (1% NP-40, 150 mM Nacl, 50 mM Tris-HCl at pH 7.4 and 2 mM EDTA) in the presence of 1X complete mini, EDTA-free protease inhibitors (Roche). The lysates were resolved on 8% SDS-PAGE gels followed by transfer of the contents to nitrocellulose membranes and immunoblotted with for 1 h with anti-FLAG antibody-peroxidase conjugate from Sigma (Panel A) or anti-HA antibody- peroxidase conjugate from Roche (Panel B). Membranes were washed 5 times with 0.05% T-TBS (0.5 ml Tween-20 in 1 L 1X TBS [20 mM Tris-HCl at pH 7.5 and 175 mM NaCl]). The peroxidase was detected using BM Blue POD substrate (Roche). Lane1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane2: Mock transfected cells; Lanes 3-5: pCMV-SHEV-transfected cells harvested at 12, 24, and 48 hrs post-transfection (the 185 kDa ORF1 translation product is denoted on the right margin). As a control, the nonstructural protein ORF (NS-ORF) of rubella virus was amplified from Robo502, an infectious cDNA clone (18), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF and cloned into VR1012 plasmid vector; the resulting construct was termed pCMV-NS-ORF. 293T cells were transfected with 5 μg of pCMV-NS-ORF DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 24 and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. Western blotting and probing of lysates was done as described in the legend to Figure 2. Lane 1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane 2: Mock transfected cells; Lanes 3-4: pCMV-NS-ORF-transfected cells harvested at 24 and 48 hrs post-transfection. The 240 kDa precursor and the P150 (N-terminal) and P90 (C-terminal) products are denoted in the right margin.

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