Franny Jongbloed

108 CHAPTER 4 A B Slot Dietary intervention Mr Marker lane 1 Control 2 CHO-free 3 Fat-free 4 Protein-free 5 3-day 30% DR 6 2-week 30% DR 7 SDS control 8 3-day fasting 9 Reference sample Slot Dietary intervention Mr Marker lane 1 Control 2 CHO-free 3 Fat-free 4 Protein-free 5 3-day 30% DR 6 2-week 30% DR 7 SDS control 8 3-day fasting 9 Reference sample Figure S5. RepresentativeWesternblots of kidney extracts for bothphosphorylated (A) and total (B) ribosomal protein S6 with β-actin as a loading control used for the relative quantification in Figure 4. The depicted exemplary blots show a full set of kidneys extracts from each indicated diet group (lanes 1 – 8) next to a marker lane (lane Mr) and a reference sample (lane 9). In total, four such sets of Western blots were generated, each with four unique complete series for each diet group. For interblot comparison the exact same amount of the reference sample (lane 9) was loaded on each blot. The relative phosphorylation ratios depicted in Figure 4. are derived from the quantified signal of the main band in each lane, subtracted with background and corrected for both the sample dependent β-actin signal and the blot dependent signal of the reference sample.