Computational protocol: MORG1+/− mice are protected from histological renal damage and inflammation in a murine model of endotoxemia

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Protocol publication

[…] For protein analyses the kidneys were extracted 24 h following LPS i.p. application or saline solution and the protein expression was evaluated. Assessment of protein was performed in kidneys homogenised in complete lysis M supplemented with inhibitors (Roche, Mannheim, Germany) and 1 mM sodium-orthovanadate. Routinely 3 to 4 animals per group were randomly selected for Western blot analyses. The lysates were vortexed, kept on ice for 15 min and centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatant was then used for the further analysis. To evaluate the protein expression 12% or 15% SDS-PAGE were performed, followed by transfer of the gels onto a PVDF membrane. The detection of the protein was performed by ImageQuantTM LAS 4000 biomolecular imager system (GE Healthcare, Upsala, Sweden) via ECL- visualisation. The following antibodies were used for protein detection: anti-phospho Iκ-Bα (1: 200 dilution), anti- Iκ-Bα (1: 200 dilution), anti-phospho-IKKα,β (Ser 180, Ser181) (1: 200), anti-IKKα (1: 200), anti-vinculin (1: 2000 dilution), anti-TNFα (1: 200 dilution), anti-PCNA (proliferating cell nuclear antigen) (1: 500 dilution), anti-NF-κB (1:200). All antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The expression of caspase-3 was detected by anti-caspase-3 antibody (1:1000 dilution) purchased from abcam, Cambridge, UK. The assessment of the nuclear NF-κB levels was performed by preparation of kidney nuclear extracts by the use of ProteoExtract® Subcellular Proteome Extraction kit (Merck, Darmstadt, Germany). The expression of the proteins was subjected to densitometry measurement using ImageJ software. [...] All values are presented as mean ± standard error of mean (SEM). Statistical analyses were performed with the statistical package SigmaPlot 13 software (SYSTAT Software, San Jose, CA, USA). Results were evaluated by the Kruskal-Wallis One way Analyses of Variances on Ranks test, followed by the Mann-Whitney-Rank Sum-Test to analyse the differences between two groups. All Pairwise Multiple Comparison Procedures were performed with Student-Newman-Keuls Method. Survival was analysed by the Kaplan-Meier test with log-rank statistic. Differences were considered significant when p < 0.05. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Kidney Diseases
Chemicals Creatinine, Periodic Acid