ace2 expression lentiviral vector  (Addgene inc)

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: ( A ) Immunoblot analysis of lysates prepared from MRC-5-ACE2 human cells that were mock-infected or infected with SARS-CoV-2 at multiplicity of infection (MOI) of 5 for 24 or 48 hr. The immunoblot was probed with anti-TRMT1, actin, or SARS-CoV-2 nucleocapsid (N) antibodies. Circle represents endogenous full-length TRMT1. Asterisk (*) denotes a non-specific band. Size markers are noted in kiloDalton. ( B ) Quantification of TRMT1 signal intensity normalized to actin in the mock or SARS-CoV-2-infected cell lines. TRMT1 protein levels are expressed relative to mock-infected samples for each time point. ( C ) m2,2G levels in small RNAs isolated from MRC5 cells that were either mock-infected or infected with SARS-CoV-2 at MOI of 5 for 24 or 48 hr. m2,2G levels were normalized to A, C, G, and U. Samples were measured in biological replicates. Statistical significance for ( B ) and ( C ) was determined by two-way ANOVA with multiple comparisons test. ***p<0.001; ****p<0.0001; ns, non-significant. ( D ) Levels of the indicated RNA modifications in small RNAs isolated from MRC5 cells that were either mock-infected or infected with SARS-CoV-2 for 24 or 48 hr. RNA modification levels were normalized to A, C, G, and U. Y-axis represents the log2 fold change in the levels of the indicated tRNA modification between SARS-CoV-2 infected versus mock-infected MRC5 cells. The experiment in ( C ) was repeated as an independent biological replicate in . Figure 1—source data 1. Raw uncropped immunoblots for . Figure 1—source data 2. LC-MS measurements of RNA modifications.

Article Snippet: For infections, the cells were previously transduced with a Lentiviral vector expressing ACE2 using the lentiviral construct RRL.sin.cPPT.SFFV/Ace2.WPRE (MT136) was kindly provided by Caroline Goujon (Addgene plasmid # 145842) ( ).

Techniques: Western Blot, Infection, Isolation, RNA modification, Modification, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: ( A ) Immunoblot analysis of lysates prepared from the indicated 293T cell lines expressing empty vector or ACE2. The immunoblot was probed with anti-ACE2 and actin. ( B ) Immunoblot analysis of lysates prepared from 293T-ACE2 cell lines that were mock-infected (multiplicity of infection, MOI 0) or infected with SARS-CoV-2 at the indicated MOI. The blot was probed against the SARS-CoV-2 nucleocapsid ( N ) and actin. Figure 6—figure supplement 1—source data 1. Raw uncropped immunoblots for .

Article Snippet: For infections, the cells were previously transduced with a Lentiviral vector expressing ACE2 using the lentiviral construct RRL.sin.cPPT.SFFV/Ace2.WPRE (MT136) was kindly provided by Caroline Goujon (Addgene plasmid # 145842) ( ).

Techniques: Western Blot, Expressing, Plasmid Preparation, Infection

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: Immunoblot of lysates from human cells integrated with empty lentiviral vector or lentiviral expression vectors for wild-type (WT) TRMT1 or TRMT1-Q530N. The cell lines were transfected with either a vector or a construct expressing Nsp5-Strep. The immunoblot was probed with anti-TRMT1, Strep, and actin antibodies. Square represents TRMT1-FLAG, circle represent endogenous TRMT1, Asterisk (*) denotes a non-specific band, and arrow represents N-terminal TRMT1 cleavage product. Figure 7—figure supplement 1—source data 1. Raw uncropped immunoblots for .

Article Snippet: For infections, the cells were previously transduced with a Lentiviral vector expressing ACE2 using the lentiviral construct RRL.sin.cPPT.SFFV/Ace2.WPRE (MT136) was kindly provided by Caroline Goujon (Addgene plasmid # 145842) ( ).

Techniques: Western Blot, Plasmid Preparation, Expressing, Transfection, Construct

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: The immunoblot was probed with anti-ACE2 and actin. Figure 7—figure supplement 2—source data 1. Raw uncropped immunoblots for .

Article Snippet: For infections, the cells were previously transduced with a Lentiviral vector expressing ACE2 using the lentiviral construct RRL.sin.cPPT.SFFV/Ace2.WPRE (MT136) was kindly provided by Caroline Goujon (Addgene plasmid # 145842) ( ).

Techniques: Western Blot

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet:

Article Snippet: For infections, the cells were previously transduced with a Lentiviral vector expressing ACE2 using the lentiviral construct RRL.sin.cPPT.SFFV/Ace2.WPRE (MT136) was kindly provided by Caroline Goujon (Addgene plasmid # 145842) ( ).

Techniques: Virus, Software, Recombinant, Plasmid Preparation, Expressing, Variant Assay, Suspension, Protein Purification, Western Blot, Imaging, Sequencing