Stress-induced phospho-ubiquitin formation causes parkin degradation

Lyudmila Kovalchuke; Eugene V. Mosharov; Oren A. Levy; Lloyd A. Greene

Membranes were imaged using an Li-Cor Odyssey CLX scanner and band intensities were quantified using Image Studio Lite software (ver. 4.0.21). Briefly, after background subtraction, the signal from the lane in which a half volume of sample was loaded was assigned a value of "0.5" using the "concentration standard" feature. Similarly, the signal from the lane corresponding to full volume of sample was assigned a value of "1". The program's linear interpolation feature was then used to assign values to the remaining bands on the blot. This process was carried out both for proteins of interest and for loading controls, after with the former were normalized to the latter. For most experiments, bands of interest were normalized to the average of the corresponding ERK1 and ERK2 signals, though sometimes Ponceau stain was also used as a loading control.