nti eif2 α  (Santa Cruz Biotechnology)

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 2. Influence of mRNA codon usage bias on sensitivity to NSP1. ( A ) Correlation between tAIg of reporter mRNAs and their corresponding sensitivities to NSP1 WT or M2. Data were scored as described in Fig. 1 . The R 2 and P -values are indicated. ( B ) Effect of RL CDS recoding on sensitivity to NSP1 WT and M2. The CAI, tAIg, and CSCg values for parental (RL) and RL-1 to three variants are indicated. Right panel shows a relative quantification of RL and RL-3 mRNA le v els in HEK293T cells expressing NSP1 WT or M2. ( C ) Correlation between TE, tAIg, and sensitivity to NSP1 WT and M2. Upper left panel shows TE calculated for the indicated mRNAs by polysome profiling followed of RT-qPCR. The resulting heat map of the relative quantification is shown. Correlation between TE and tAIg (upper right panel) or TE and sensitivity to mRNA-induced degradation by NSP1 WT or M2 (bottom panel). Data were scored as described above and the resulting R 2 is indicated. Bottom right panel shows the correlation between TE of human mRNAs and sensitivity to NSP1-induced mRNA degradation in Calu3 cells infected with SARS-CoV-2 WT compared with that infected by a mutant virus defective in NSP1 ( NSP1). Data were taken from GSE200422 [ 27 ] and analyzed as described in the ‘Materials and methods’ section; the resulting Spearman correlation coefficient (r) is indicated. ( D ) Effect of SARS-CoV-2 infection on expression of RL-3, FFL, and EGFP in VeroE6 cells. Cells were infected, transfected with a combination of the three plasmids and analyzed at 16 and 24 hpi for luciferase activity (upper panel) or by western blot against NSP1, RPS6, and EGFP. In parallel wells, cells were co-transfected with pNSP1 and a combination of RL-3, FFL, and EGFP plasmids and analyzed 24 h later (top panels). A comparative analysis of NSP1 levels accumulated in infected and transfected cells is shown (bottom panel). Blots were also probed with anti-eIF2 α as loading control. The 1 / 2 and 1 / 4 dilutions of cell extracts are also indicated.

Article Snippet: In both cases, a standard curve was included for elative quantification in the linear range of measurement. estern blot estern blots were carried out as described previously 14 , 26 ] using the following primary antibodies: anti-EGFP 11 814 460 001, Roche), anti-dsRed (a gift from José aría Requena, CBM, Madrid.), anti-FFLuc (a gift from raceli del Arco, CBM, Madrid), anti-N S AR S-CoV-2 (MA58034, Invitrogen), anti-NSP1 S AR S-CoV-2 (STJ11103222, t. John’s lab), anti-myc (05-724MG, Merck-Millipore), antiIF4A (STJ2724, St. John’s lab), and anti-eIF3G (STJ23512, t. John’s lab), anti-ABCE1 (STJ11102433, St. John’s lab), and nti-eIF2 α (sc-11386, Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Infection, Mutagenesis, Virus, Transfection, Luciferase, Activity Assay, Western Blot, Control