All transfected cells were exposed to G418 (800 μg/mL, Sigma Chemical Co., St. Louis, USA) for 3 weeks of selection. Resistant clones representing stably transfected cells were ring-cloned and expanded for further experiment. siRNAs against EGFR were transfected into T24 and Selleckchem Bafilomycin A1 5637 cells according to the transfection protocol of Lipofectamine2000

(Invitrogen). A nonspecific control siRNA strand was used as a negative control. Seventy-two hours after transfection, knockdown was assessed by western blot from a parallel transfection. After downregulation of EGFR, we detected the effect of LRIG1 cDNA on cell proliferation and EGFR signaling pathway by CCK-8 assays and western blot respectively. Quantitative real-time RT-PCR Total RNA was extracted from 45 cases of bladder cancer and 5 cases of respective non-neoplastic tissue samples and 2 bladder cancer cell lines with Trizol reagent. The expression of LIG1 and EGFR mRNA was done using quantitative real-time RT-PCR. RNA samples were run in triplicate using 20 ng of RNA perreaction. The resulting cDNA samples were amplified by real-time PCR using gene-specific primer sets in conjunction with the SYBR Premix Ex Taq (TaKaRa) in a Mx3000p instrument. The qPCR was performed with the following conditions: activation at 95°C for 5 min followed by 40 cycles of denaturation at 94°C for 15 s, amplification at 60°C for 30 s, elongation at 72°C for 30 s. In the

last, a cycle of solubility curve was added to examine the amplification quality. Expression of mRNA for GAPDH was used as an internal Microtubule Associated inhibitor standard. Reverse transcription products were amplified

by PCR using specific primers for human LRIG1 (forward 5′-GGTGAGCCTGGCCTTATGTGAATA-3′; reverse 5′-GGTGAGCCTGGCCT TATGTGAATA-3′) and human EGFR (forward 5′-TCCCTCAGCCACCCATAT GTAC-3′; reverse 5′-TCCCTCAGCCACCCATATGTAC-3′). Immunohistochemistry(IHC) Formalin-fixed and paraffin-embedded tissue sections (5 mm) were dewaxed with xylene and rehydrated through an ethanol gradient into water. Following blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide for 10 min, the sections were washed with phosphate buffered saline(PBS) and incubated over-night with rabbit LRIG1 antibody or EGFR antibody at the dilution of 1:100 in a humidified chamber at 4°C. After 4-Aminobutyrate aminotransferase washing with PBS, sections were incubated with biotinylated secondary antibody for 30 min at 37°C and then with horseradish peroxidase labeled streptavidin for 30 min at 37°C. Diaminobenzidine(DAB) was used as chromogen and the sections were subsequently counterstained with hematoxylin, then dehydrated, cleared and mounted. Western blotting analysis The transfected bladder cancer cells were collected and washed with 0.01 mol/L PBS for three times. Then the cells were added into 200ul pre-cold RIPA-PICT cell disruption liquor and centrifuged. All subsequent manipulations were performed on ice. After centrifugation, the supernatant was collected.