Journal: Biochemical and Biophysical Research Communications

Article Title: VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability

doi: 10.1016/j.bbrc.2015.02.097

Figure Lengend Snippet: VHL interacts with SARS-CoV nsp16. (A) 6xHis-VHL or 6xHis-nsp16 proteins expressed in BL21 cells were incubated with glutathione beads and the purified GST, GST-nsp16 or GST-VHL proteins. The bound proteins were eluted and detected by Western blotting with an anti-His or anti-GST antibody. (B) Flag-nsp16 and HA-VHL were transfected into HEK293T cells. After 36 h, the cell extracts were immunoprecipitated (IP) with an anti-HA or anti-Flag antibody and analyzed by immunoblotting (IB) with an anti-Flag or anti-HA antibody (top panel). (C) The eGFP-nsp16 and Red-VHL plasmids were co-transfected into HeLa cells. After 24 h, the cells were fixed with 4% paraformaldehyde and the nuclei were stained with DAPI. The cells were examined by confocal microscopy. (D) After transfection, the Vero cells were fixed with 4% paraformaldehyde, labeled with rabbit anti-Flag antibody (1:100), then probed with FITC-tagged goat anti-rabbit IgG. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The mouse anti-Flag (F4045) and anti-HA (H9658), the rabbit anti-Flag (F7425) antibodies were purchased from Sigma–Aldrich.

Techniques: Incubation, Purification, Western Blot, Transfection, Immunoprecipitation, Staining, Confocal Microscopy, Labeling

Journal: Biochemical and Biophysical Research Communications

Article Title: VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability

doi: 10.1016/j.bbrc.2015.02.097

Figure Lengend Snippet: VHL regulates SARS nsp16 protein stability, degradation, and SARS replication. (A) Flag-nsp16 and Flag-VHL plasmids were transfected into HEK293T cells. After 24 h, the cells were treated with CHX (100 μg/ml) for the different times. The cell lysates were analyzed by Western blotting with an anti-Flag or anti-β-actin antibody. (B) Flag-nsp16 and HA-VHL plasmids were transfected into HEK293T cells. The cells were treated with MG132 (20 μM) or NH 4 Cl (10 mM) for another 6 h after 24 h transfection. The cell lysates were analyzed by Western blotting with different antibodies. (C) HEK293T cells were transfected with Flag-nsp16 and control (PLKO.1) or VHL knockdown plasmids (sh-VHL-1#, sh-VHL-2#). After 24 h, the cells were treated with puromycin (1 μg/ml) for an additional 16 h. The cell lysates were analyzed by Western blotting with indicated antibodies. (D) The SARS-rep-LUC (0.1 μg) and HA-VHL (0.3 μg) were transfected into Vero cells. The pcDNA3.0 and pRL-TK were used to make an equal amount of total DNA and normalize the transfection efficiency. The reporter assays were processed 36 h after transfection. (E) Assays were performed as in (D) except for the transfection with VHL RNAi plasmids. *, p < 0.05, **, p < 0.01; the bar graphs show the mean value ± SD of triplicate samples.

Article Snippet: The mouse anti-Flag (F4045) and anti-HA (H9658), the rabbit anti-Flag (F7425) antibodies were purchased from Sigma–Aldrich.

Techniques: Transfection, Western Blot

Journal: Biochemical and Biophysical Research Communications

Article Title: VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability

doi: 10.1016/j.bbrc.2015.02.097

Figure Lengend Snippet: VHL modulates SARS-CoV nsp16 ubiquitination. (A) Left panels, HEK-293T cells were transfected with Myc-ubiquitin, Flag-nsp16 and an increasing amount of HA-VHL (1.0 and 2.0 μg). After 36 h transfection, the cells were collected and denatured in 1% SDS lysis buffer, and the cell lysate were diluted with lysis buffer until the concentration of SDS decreased to 0.1%. The cell extracts were used for co-immunoprecipitation with an anti-Flag antibody. The immunoprecipitates were detected by an anti-Myc or anti-Flag antibody. Right panels, assays were performed as in the left panels. SARS-CoV nsp14 as a negative control and Dvl2 as a positive control. (B) The HA-tagged ubiquitin mutants, Flag-nsp16 and Myc-VHL were transfected into HEK-293T cells. After 36 h, re-IP assays were performed with anti-Flag antibody. The immunoprecipitates were analyzed with an anti-HA or anti-Flag antibody. (C) The HA-ubiquitin, Flag-nsp16 or Flag-nsp14 or Flag-Dvl2, control (PLKO.1) or VHL RNAi plasmids (1# or 2#) were transfected into HEK-293T cells. After 24 h, the cells were treated with puromycin (1 μg/ml) for another 24 h and then lysed for re-IP assays with anti-Flag antibody. The immunoprecipitates were analysed with the anti-HA or anti-Flag antibody (upper panels).

Article Snippet: The mouse anti-Flag (F4045) and anti-HA (H9658), the rabbit anti-Flag (F7425) antibodies were purchased from Sigma–Aldrich.

Techniques: Transfection, Lysis, Concentration Assay, Immunoprecipitation, Negative Control, Positive Control

Journal: Neural Regeneration Research

Article Title: Repairing whole facial nerve defects with xenogeneic acellular nerve grafts in rhesus monkeys

doi: 10.4103/1673-5374.324853

Figure Lengend Snippet: Morphological assessment at 8 months postoperatively . The Retrograde Labeling Test (original magnification, 400×; A, B) revealed FluoroGold-labeled neurons with multipolar dendrites in the facial nerve nuclei. Hematoxylin-eosin staining (original magnification, 200×; C, D) and S100 immunohistochemical staining (original magnification, 200×; E, F) showed the regenerated nerve fibers, and S100-positive Schwann cells and myelin sheaths were densely clustered, with different sizes, in the transverse section. Transmission electron micrographs (original magnification, 1200×; G, H) showed that most myelin sheaths were arranged in the mature and uniform lamellar pattern, and that the Schwann cells had perfect basement membranes. Synaptophysin immunohistochemical staining (original magnification, 200×; I, J) of the orbicularis oris muscle showed that some motor endplates were reinnervated in the muscle. (A, C, E, G, I) The autograft group; (B, D, F, H, J) the xenogeneic acellular nerve graft group.

Article Snippet: Immediately after electrophysiological monitoring at 8 months postoperatively, 15 μL of 5% FluoroGold solution (F4040, US EVERBRIGHT®INC., Suzhou, Jiangsu Province, China) was injected at multiple points into the buccal division of the facial nerve, distal to the nerve graft.

Techniques: Labeling, Staining, Immunohistochemical staining, Transmission Assay