Two rabbits were immunized twice with the acetyl peptide, and the sera from these rabbits was affinity purified. Two antigen-affinity columns were used to purify the acetyl-specific antibodies. The first column was the non-acetyl peptide affinity column. Antibodies recognizing the non-acetylated residues of the peptide bound to the column. The flow-through fraction was collected and then applied to the second column, the acetyl peptide column.
For immunoprecipitation studies, cell lysed by sonication in RLB, the lysates were quantitated, and 0.5 to 1 mg of protein lysate was immunoprecipitated using 2 μg of anti-SREBF1, anti-pTyr, anti-FLAG, anti-HA, anti-Myc or anti-AR antibody coupled with protein A/G sepharose overnight, followed by washes with RLB and PBS buffers. The beads were boiled in a sample buffer and immunoblotting was performed as described above.
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Systems genetics identifies miRNA-mediated regulation of host response in COVID-19 - Human GenomicsBackground Individuals infected with SARS-CoV-2 vary greatly in their disease severity, ranging from asymptomatic infection to severe disease. The regulation of gene expression is an important mechanism in the host immune response and can modulate the outcome of the disease. miRNAs play important roles in post-transcriptional regulation with consequences on downstream molecular and cellular host immune response processes. The nature and magnitude of miRNA perturbations associated with blood phenotypes and intensive care unit (ICU) admission in COVID-19 are poorly understood. Results We combined multi-omics profiling—genotyping, miRNA and RNA expression, measured at the time of hospital admission soon after the onset of COVID-19 symptoms—with phenotypes from electronic health records to understand how miRNA expression contributes to variation in disease severity in a diverse cohort of 259 unvaccinated patients in Abu Dhabi, United Arab Emirates. We analyzed 62 clinical variables and expression levels of 632 miRNAs measured at admission and identified 97 miRNAs associated with 8 blood phenotypes significantly associated with later ICU admission. Integrative miRNA-mRNA cross-correlation analysis identified multiple miRNA-mRNA-blood endophenotype associations and revealed the effect of miR-143-3p on neutrophil count mediated by the expression of its target gene BCL2. We report 168 significant cis-miRNA expression quantitative trait loci, 57 of which implicate miRNAs associated with either ICU admission or a blood endophenotype. Conclusions This systems genetics study has given rise to a genomic picture of the architecture of whole blood miRNAs in unvaccinated COVID-19 patients and pinpoints post-transcriptional regulation as a potential mechanism that impacts blood traits underlying COVID-19 severity. The results also highlight the impact of host genetic regulatory control of miRNA expression in early stages of COVID-19 disease.
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