Urothelial cells and tissues were scratched from freshly removed bladders of UPII-mutant Ha-ras mice and protein lysates were prepared through homogenization as previously described [13 (link),14 (link),16 (link)]. Amounts of 10 to 50 μg protein lysates were used for Western blotting analysis of the protein expression of TSC2, phospho-mTOR, 4E-BP1, S6, Skp2, and β-tubulin (as a loading control) using a previously established protocol [10 (link)]. All the antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA).
In addition, proliferating cells on formalin-fixed and paraffin-embedded bladder tissue sections from mice fed with normal drinking water or SHR-5-containing water were determined by IHC analysis using previously reported protocols. Anti-Ki-67 antibody (1:800) was obtained from Abcam an anti-phospho-MAPK (at Thr202/Tyr204) Antibody antibody (1:50), anti-Phospho-S6 Ribosomal Protein (rpS6) (Ser235/236), and an anti-p27/Kip1 antibody from Cell Signaling Technologies (Danvers, MA, USA) [13 (link),14 (link),16 (link)]. The quantification of the Ki67-positive cells was carried out by counting the total number of positively stained cells at 5 arbitrarily selected fields at ×200 magnification in a double-blinded manner.
In addition, proliferating cells on formalin-fixed and paraffin-embedded bladder tissue sections from mice fed with normal drinking water or SHR-5-containing water were determined by IHC analysis using previously reported protocols. Anti-Ki-67 antibody (1:800) was obtained from Abcam an anti-phospho-MAPK (at Thr202/Tyr204) Antibody antibody (1:50), anti-Phospho-S6 Ribosomal Protein (rpS6) (Ser235/236), and an anti-p27/Kip1 antibody from Cell Signaling Technologies (Danvers, MA, USA) [13 (link),14 (link),16 (link)]. The quantification of the Ki67-positive cells was carried out by counting the total number of positively stained cells at 5 arbitrarily selected fields at ×200 magnification in a double-blinded manner.