Journal:

Article Title: The Glomerulosclerosis of Aging in Females

doi:

Figure Lengend Snippet: Increased NF-κB transcriptional activity in mesangial cells isolated from 28-month-old mice. Two separate mesangial cell lines were cultured from 5-month-old (5-month MC) or 28-month-old (28-month MC) female B6 mice. NF-κB transcriptional activity was measured by transfecting cells with a luciferase NF-κB reporter construct and a β-galactosidase plasmid. Basal NF-κB transcriptional activity in 5-month MC were arbitrarily defined as 1. Data shown are from six measurements of each of four independent experiments. A: Basal NF-κB transcriptional activity (base) was 2.6-fold higher in 28-month MC than in 5-month MC. The introduction of an exogenous IKK expression vector to 5-month MC or 28-month MC increased cell NF-κB transcriptional activity. In contrast, transfection of 5-month or 28-month MC with an IκB-α or dominant-negative IKK (dn) expression vector decreased cell NF-κB transcriptional activity. B: Increased sensitivity to TNF-α induced NF-κB activation in 28-month MC. Five ng/ml of TNF-α increased NF-κB transcriptional activity in 28-month MC but not in 5-month MC. An increase in TNF-α levels to 10 ng/ml was required to stimulate NF-κB activity in 5-month MC.

Article Snippet: Transfection and NF-κB Reporter Gene Assay Transient transfection was performed in mesangial cells from 5- and 28-month-old mice using Transfast according to the manufacturer’s protocol (Promega, Madison, WI).

Techniques: Activity Assay, Isolation, Cell Culture, Luciferase, Construct, Plasmid Preparation, Expressing, Transfection, Dominant Negative Mutation, Activation Assay