Developmental and tissue specific changes of ubiquitin forms in Drosophila melanogaster
Fig 3
Effect of loss of Rpn10/p54 (A and C) proteasome subunit or Usp5 deubiquitylase (B and C) on the abundance of ubiquitin forms. Whole protein extracts in buffer F (lanes 1, 2, 5 and 6; +NEM) and buffer T (lanes 3, 4, 7 and 8; -NEM) of wandering L3 larvae were investigated by western blotting using polyclonal anti-Ub antibody at a dilution of 1:1000. The bands just below the 10 kDa mark on the immunoblots (A and B) represent free monoubiquitins, and only the intensity of these bands were determined and used for quantification. 5 μg total protein extracts were loaded to lanes 2 and 6, while samples were diluted 1.7-fold to lanes 1 and 5; twofold to lanes 3, 4 and 8; and 3.3-fold to lane 7 before loading to avoid overloading the monoubiquitin band. Ubiquitin content (small table in C) was calculated by plotting band intensities against Ub standards and a regression line equation was generated by applying the four parameter curve fit model (R2 = 0.9979 for Rpn10/p54 and R2 = 0.9933 for Usp5), values were normalized to total protein content and shown as a column diagram.