The mixture was heated to 100°C for

5 min to denature the proteins. The protein from each sample was subjected to electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gel. Then protein was transferred to nitrocellulose membrane, which were blocked with PBS containing 5% non-fat milk for 2 h and then incubated with anti-LRIG1 (1:5,000), anti-EGFR(1:2,000), anti-p-EGFR(1:2,000), anti-MAPK(1:2,000), anti-p-MAPK(1:2,000), anti-AKT(1:2,000), anti-p-AKT(1:2,000), anti-caspase-8(1:1,000), anti-MMP-2(1:2,000), anti-MMP-9(1:2,000) and β-actin(1:2,000) at 4°C overnight. Then secondary antibody labeled with alkaline selleck compound phosphatase were added at room temperature. One hour later, the samples were washed for three times with TBST, and then visualized using DAB Milciclib nmr detection system. Immunoprecipitation The total protein was prepared using M-PERTM mammalian protein extraction reagent (PI3K inhibitors ic50 Pierce). For each sample, 10 μL of anti-LRIG1 antibody or control

IgG was added to 1 mg of protein in 200 μL of lysis buffer and placed on a rocker overnight at 4°C. Twelve microliters of protein G beads was added to each sample, which was placed on a rocker at 4°C for 1 h. The beads were washed three times with 1 ml of lysis buffer and then boiled in 50 μL of SDS sample buffer; 20 μL was then loaded per lane and subjected to Western blotting. Apoptosis analysis Annexin V-PE/7-aad double staining assay was used to detect cell apoptosis. After transfected and incubated for 3 days, cells were collected, centrifuged and washed with phosphate—buffered saline(PBS) for two times. Binding

buffer was then added to each tube and cells were re-suspended. The cells were incubated with 5 μL of annexin V-PE and 5 μL of 7-aad for 15 min at room temperature in the dark. Then, the apoptotic analyses were done by flow cytometry within one hour. Survival assay by CCK-8 The growth of T24 and 5637 cells after LRIG1 gene MG-132 solubility dmso transfection were evaluated by Cell Counting Kit-8 assays. Untreated cells, cells treated with liposome alone and cells treated with the vector control were used for comparison. Cell suspensions (at 1 × 103/mL) were transferred to 96-well plates in triplicate and incubate for 24, 48 and 72 hours. Subsequently, CCK-8(10 μL) was added to each well, cells were incubated for an additional 4 h. Then, The values of each well was measured by microplate reader at 450 nm. Clonal forming assay T24 and 5637 cells were infected with LRIG1 cDNA and cultured for 24 h, then plated in 6-well plates at 200 cells/well. Plates were subsequently incubated for 14 days in a humidified incubator at 37°C, and the colonies were stained with 0.5 ml of 0.0005% crystal violet solution for 1 h and counted by using a microscope. Five random fields were counted from each sample and average values presented ± the SD. Matrigel invasion assays The in vitro invasive ability of bladder cancer cells was measured in transwells chambers assay.