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Muscle histology, biochemical and molecular analyses of anabolic/catabolic and inflammatory signalling pathways were performed.
Statistical analyses for individual genes and pathways were performed on 1719 cell adhesion-related genes (Gene Ontology database, http://www.geneontology.org) as well as the Chip-wise genes.
A statistical enrichment analysis of functional categories and pathways were performed using Ingenuity Pathway Analysis system IPA 6.0 (Ingenuity Systems, www.ingenuity.com).com
QRT-PCR analysis of select genes present in gene networks and affected pathways were performed including GSTM4, NRF2, ALDH1A1, and fatty acid binding protein 2 (FABP2) had R2 values of 0.85, 0.83, 0.73, and 0.72, respectively, when compared to microarray data across all samples (File S1).
Given the involvement of INT6 in cell proliferation [16], western blots using a panel of antibodies against proteins involved in the cell cycle and associated signalling pathways were performed using lysates from control and INT6 siRNA transfected MDA-MB-231 cells.
The CD44 protein-protein interaction pathways were performed by String 9.0 Web software.
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Enrichment of pathways was performed by DAVID (v6.8) analysis with the corresponding groups of differentially expressed genes.
Further investigation of gene functional pathways was performed using Gene Set Enrichment Analysis (http://www.broad.mit.edu/gsea/) as described67.
Enrichment analysis of pathways was performed using Fisher's Exact Test.
Gene enrichment in pathways was performed at the DAVID web server [41] using a P-value of 0.5, interrogating KEGG database.
Hierarchical average linkage clustering of data for selected pathways was performed using MeV v4.2 [101] with Euclidean and Pearson correlation as distance metrics for completeness and activity scores, respectively.
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