sino biological catalog 50385 mnac  (Sino Biological)

Journal: Science Advances

Article Title: Conserved sites on the influenza H1 and H3 hemagglutinin recognized by human antibodies

doi: 10.1126/sciadv.adu9140

Figure Lengend Snippet: ( A ) Diversity of influenza A HA. Phylogenetic tree of IAV HAs (left). Timeline of influenza H1N1 and H3N2 circulation; symbols represent strains used in this study (middle). Pairwise identity of HAs from viruses used in this study mapped onto structures of HA [Protein Data Bank (PDB) 3LZG and 4FNK] (right). ( B ) Neutralization potencies determined by microneutralization for down-selected antibodies against a panel of IAVs. Strains are indicated as in (A). H1N1 strains: A/WSN/1933, A/USSR/90/1977, A/Memphis/4/1987, A/Massachusetts/1/1990, A/Beijing/262/1995, A/New Caledonia/20/1999, A/Solomon Islands/3/2006, A/New York/08-1326/2008, A/California/07/2009, A/Michigan/45/2015, and A/Sydney/5/2021. H3N2 strains: A/Aichi/2/1968, A/Bilthoven/1761/1976, A/Beijing/353/1989, A/Johannesburg/33/1994, A/Brisbane/8/1996, A/Fujian/411/2002, A/Wisconsin/67/2005, A/Perth/16/2009, A/Switzerland/9715293/2013, and A/Kansas/14/2017. Neutralization assays were performed in technical duplicate. N.N., non-neutralizing. ( C ) ELISA binding reactivity at 10 or 1 μg/ml to A/Swine/Henan/SN13/2018 or A/Canine/Hainan/79/2019 (left) and BLI-based antibody competition with the indicated antibodies (right).

Article Snippet: Briefly, His-tagged H1N1 A/California/04/2009 full-length trimer Y97F (Sino Biological, catalog no. 11055-V08B1) was mixed in 4:1 molar ratios with SA-allophycocyanin (APC; Invitrogen) and SA-phycoerythrin (PE; Invitrogen) whereas A/Darwin/6/2021 full-length soluble trimer and A/Hong Kong/1/1968 head trimer (see below) were mixed in 4:1 molar ratios with SA-PE (Invitrogen) and SA-APC (Invitrogen), respectively, and were incubated for 20 min on ice.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: bioRxiv

Article Title: Extracellular ISG15 triggers ISGylation via a type-I interferon independent mechanism to regulate host response during virus infection

doi: 10.1101/2024.07.05.602290

Figure Lengend Snippet: A) Human lung epithelial Calu-3 cells were infected with SARS-CoV-2 (MOI= 0.1). Whole cell lysate (WCL) collected at 48h post-infection (hpi) and 120hpi was subjected to immunoblotting with ISG15, PLpro and actin antibodies. B) Cell culture supernatants (supernatant) from SARS-CoV-2 (MOI= 0.1) infected Calu-3 cells were collected at 120 hours post infection. Trichloroacetic acid (TCA) precipitated supernatant was subjected to immunoblot with anti-ISG15 antibody to detect extracellular ISG15. C) Supernatants from mock and SARS-CoV-2 infected (120 hpi) Calu-3 cells were used to quantify cell death by lactate dehydrogenase (LDH) assay. D) Experimental schematic – human lung epithelial A549 cells were transfected with either plasmids encoding C9-tagged S proteins from various coronaviruses or empty vector control plasmid pcDNA 3.1 (pcDNA). At 24h post-transfection, washed cells were incubated with fresh media without plasmids for 16h and 24h. Whole cell lysate (WCL) was collected for analysis. E) WCL collected from A549 cells transfected with C9-tagged SARS-CoV-2 (SARS2) S protein as described in the experimental schematic in was subjected to immunoblotting with ISG15, C9, and actin antibodies. A549 cells treated with interferon-β (IFN-β, 100U/mL) for 24 was used as a positive control for ISGylation. F) A549 cells were transfected with C9-tagged S protein from various coronaviruses including MERS-CoV (MERS), 229E, SARS-CoV-2 (SARS2) and SARS-CoV-1 (SARS1) or empty vector control (pcDNA). The experiment was conducted as described in the experimental scheme shown in . WCL collected at 24h was subjected to immunoblotting with ISG15, C9, and actin antibodies. G) Experimental schematic - A549 cells were transfected with either plasmid encoding C9-tagged SARS-CoV-2 S protein or empty vector control (pcDNA). At 24h post-transfection, washed cells were incubated with fresh media without plasmids for 8h, 16h and 24h. Total protein in plasmid-free culture supernatant was precipitated with TCA. H) TCA precipitated culture supernatant collected from A549 cells transfected with C9-tagged SARS-CoV-2 (SARS2) S protein as described in the experimental schematic in was subjected to immunoblotting with ISG15 antibody to detect extracellularly released ISG15.

Article Snippet: Cells were treated with 500 U/ml of IFN-β (Sino Biological, catalog no. 10704-HNAS) or vehicle prepared in antibiotic free DMEM supplemented with 10% FBS.

Techniques: Infection, Western Blot, Cell Culture, Lactate Dehydrogenase Assay, Transfection, Plasmid Preparation, Control, Incubation, Positive Control