Journal: Nature
Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance
doi: 10.1038/s41586-024-08471-0
Figure Lengend Snippet: a , HEK293-CD13-myc ISG15 stable cells were infected with HCoV-229E at an MOI of 0.01 for 72 h prior to collection of cell lysates (lysates) and supernatant (S/N) prior to western blot with anti-myc Ab (as above) to detect transfected ISG15. The level of ISG15 in the supernatant was normalized to cell lysates and loading control (GAPDH), and was expressed relative to the most abundant protein in the supernatant ( Mops condylurus ). Only ISG15 of Mops condylurus was significantly secreted into the supernatant and secretion further increased during HCoV-229E infection. Mean and standard error of the mean are displayed, including individual points (black) for n = 3 independent experiments. Significance was tested with a two-tailed t-test comparing infection to ISG15 alone. Raw data are provided in Supplementary Table . b , An example western blot image (as per panel a) showing high and low exposure of ISG15 supernatant (S/N), HCoV-229E N protein, GAPDH and ISG15 bands in the HEK293-subclone-ΔISG15 cell lysate with protein ladders. Lanes are as indicated. c , Example western blot image with low and high contrast showing ISG15 conjugation (α-myc) after transfection with ISG15’s and HCoV-229E infection (−/+). Arrows indicate bands not seen in the empty vector control (conjugated proteins). d , Western blot of E1 ligase (UBE1L), E2 ligase (UBE2L6) and E3 ligase (HERC5) expression, together with GAPDH in the same samples shown in panel c, indicating sufficient expression of ISGylation machinery in the HEK293-subclone-ΔISG15 cell line. e , HEK293-subclone-ΔISG15 cells were transfected with ISG15 constructs as indicated and with/without NSP3C/L (SARS-CoV-2 PLpro). As per panel c, arrows indicate ISGylation bands, or ISG15 dimer/monomer. The amount of ISG15 present was calculated, normalized to GAPDH and quantified from three western blots. One of the three western blots is shown as an example. f , Representative western blot for the amount of ISG15 present in the supernatant (matched to e). g , ISGylation machinery expression, as per panel d, for NSP3C samples used in panel e and f and Fig. . Western blots in panels b-g are matched to the quantification in Fig. .
Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).
Techniques: Infection, Western Blot, Transfection, Control, Two Tailed Test, Conjugation Assay, Plasmid Preparation, Expressing, Construct
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![Fig. 4 <t>HERC5</t> enhances the stability of SNAI1 mRNA by inducing UGDH phosphorylation. (A). Protein interaction network of human HERC5, as determined by BioGRID. Many HERC5-interacting proteins, including ISG15 and UGDH, were identified. (B). Schematic illustration of UGDH to promote EMT by increas ing the stability of SNAI1 mRNA, as reported by Nature. 2019;571:127–131 [18]. UGDH, UDP-glucose 6-dehydrogenase. HuR, Hu antigen R. UDP-Glc, uridine diphosphate-glucose. UDP-GlcUA, UDP-glucuronic acid. (C). UGDH protein abundance, phosphorylation level at tyrosine 473, and ratio of Phospho.Y473/ Total UGDH in head and neck cancer (HNC) and normal paracancerous tissues by Cancer Proteogenomic Data Analysis Site (cProSite). (D). Immunoprecipita tion of tyrosine-phosphorylated proteins in lysates from OSCC and para-OSCC tissues, using a pan-phospho-Tyrosine antibody. Western blot analysis was performed using an anti-UGDH antibody to assess tyrosine phosphorylation levels of UGDH in OSCC vs. para-OSCC tissues. (E). SNAI1 mRNA expression lev els in 8 paired OSCC and para-OSCC tissues of our independent hospital cohorts. (F). SNAI1 protein expression levels in 8 paired OSCC and para-OSCC tissues from our independent hospital cohorts. P, para-OSCC tissues. T, OSCC tissues. (G) Relative mRNA levels of SNAI1 in SCC9 (left) or CAL27 (right) cells. (H) SCC9 (left) or CAL27 (right) cells were treated with actinomycin D (1 μg/ml). Remained SNAI1 mRNA levels after treatment were examined at the indicated time points. (I). Immunoprecipitation of tyrosine-phosphorylated proteins in lysates from SCC9 (left) or CAL27 (right) cells, using an anti-pan-phospho-Tyrosine antibody. Western blot analysis was performed using an anti-UGDH antibody to assess tyrosine phosphorylation levels of UGDH. (J) Remained SNAI1 mRNA levels in SCC9 cells after treatment were examined at the indicated time points. (K). SCC9 cells stably overexpressing HERC5 were transiently transfected with pcDNA 3.1 (+) plasmids of a C-terminally Flag-tagged UGDH (wild type or Tyr473Phe (Y473F) mutant type). After 48 h, the remaining SNAI1 mRNA levels were examined at the indicated time points. Data in (G, H, J, K) were expressed as mean values ± SD. #/*/& p < 0.05, ##/**/&& p < 0.01 and ###/***/&&& p < 0.001](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_5362/pm40045362/pm40045362__page11_image1.jpg)
