Fig 1.
An immunoblot based assay to quantitate free and total ubiquitin content of whole protein extracts.
In this blot, parallel pupal samples (from stage P4 pupae) homogenized in buffers F (lanes 1 and 2; +NEM) and buffer T (lanes 3 and 4; -NEM) were loaded and polyclonal anti-Ub antibody at a dilution of 1:1000 was used to detect ubiquitin. Only the intensity of the free monoubiquitin band (just below the 10 kDa mark) was determined and used for quantification. 5 μg total protein extracts were loaded to lanes 1 and 2, while samples were diluted twofold before loading to lanes 3 and 4 to avoid overloading the monoubiquitin band. Ub standards of 0.5, 1, 2 and 3 pmol were loaded to lane 5–8 respectively, for the calibration curve. The inset shows the calibration curve that was used to calculate Ub concentrations in these samples. Band intensities were plotted against Ub standards and a regression line equation was generated by applying the four parameter curve fit model (R2 = 0.9978).
Fig 2.
Developmental (A) and tissue specific (B) profile of free (light blue) and total (dark blue) ubiquitins and free/total ubiquitin ratios (C and above columns in B). The colour bar at the bottom indicates the length of the developmental stages at 25°C. For panel B, tissues were prepared from third instar larvae (L3) and three day old adults. Ubiquitin content was normalized to total protein content. Data are presented as mean±SD (see also S1 Table) of three independent experiments (n = 3). The data were compared using one-way analysis of variance (ANOVA) followed by SNK post-hoc test (see S2 Table).
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.