non-linear fit one phase exponential decay curves  (GraphPad Software Inc)

Journal: PLoS Genetics

Article Title: The Rts1 Regulatory Subunit of Protein Phosphatase 2A Is Required for Control of G1 Cyclin Transcription and Nutrient Modulation of Cell Size

doi: 10.1371/journal.pgen.1000727

Figure Lengend Snippet: (A) Inactivation of the SCF E3 ubiquitin ligase does not cause increased Cln2 protein levels in rts1 Δ cells. Cells of the indicated genotypes were released from an α factor arrest into pre-warmed media at 37°C. Levels of Cln2-3XHA protein were monitored by Western blotting. (B) rts1 Δ does not decrease the half-life of the Cln2 protein. GAL1-3XHA-CLN2 and GAL1-3XHA-CLN2 rts1 Δ cells were grown overnight at room temperature in YEP media containing 2% glycerol/ethanol. A burst of CLN2 transcription was triggered by washing the cells into YEP +2% galactose for 1 hour. At time = 0, CLN2 transcription was turned off by washing cells into YEPD media. Samples were collected at t = 0, 5, 10, 15, 20, 30, 45, 60, and 120 minutes, and quantitative Western blotting was used to determine Cln2 levels. The half-life of Cln2 protein was calculated using a standard curve fitted for one phase exponential decay. The half-life of Cln2 was 9.6±2.0 minutes in wild type cells and 10.8±2.4 minutes in rts1 Δ cells (n = 3).

Article Snippet: Non-linear regression curve fitting for one phase exponential decay was carried out using GraphPad Prism version 4.00 for Mac .

Techniques: Ubiquitin Proteomics, Western Blot