The curcumin-induced apoptosis was also analyzed by Western blot using anti-PARP antibody

reased expression of KLK5 and KLK7 protein in EBS-DM cell lines determined in 48-h-conditioned cell culture supernatant by western blot. Student’s t-test was BQ-123 web performed with p values: 0.01, 0.005. doi:10.1371/journal.pone.0070123.g001 Kit, BIO-RAD, 170-8891) were also performed following the manufacturers’ protocols. Primers were designed to bind over exon-exon junctions to exclude binding to intronic sequences and to amplify an equal product length of 150 bp for all target genes. GAPDH was used as a reference gene. SQRT-PCR was performed using iQ SYBR Green Supermix in a BIO-RAD CFX96TM Real-Time System, C1000TM Thermal Cycler. A three-step protocol was used, and the 22DDCt method was applied for quantification of gene expression. All SQRTPCR results are given in fold expression. The secondary antibodies were then applied to the membrane containing the samples as well as to the negative control. BIO-RAD Precision ProteinTM StrepTactin-HRP conjugate was applied to the size marker. The secondary antibodies were incubated for 1 h at room temperature and the membrane was then washed three times for ten minutes with TBS containing 0.2% Tween-20. HRP staining solution was prepared 1:1 and applied to the membrane as well as to the negative control and to the size marker. The membrane was placed between two layers of transparent 26507655 foil and analyzed on a BIO-RAD Molecular ImagerH ChemiDocTM XRS system using Quantity one 4.6.5 software. Immunoprecipitation of Active Cdc42 The evening before the experiment 96105 cells/well of every cell line were seeded into 6-well plates and incubated in RM medium over night at 37uC, 5% CO2 in a humidified atmosphere. All of the following steps were performed on ice or at 4uC. The next day, the medium was discarded and the cells were washed once with ice-cold PBS. 550 ml lysis buffer were applied per well and the cells were scraped off using a rubber policeman. The 6-well plates were kept on ice during the procedure. The lysates were transferred to 1.5-ml reaction tubes and incubated for 45 minutes at 4uC with constant rotation. The tubes were centrifuged at 15,000 g for 10 minutes at 4uC. The supernatant, containing the cleared lysate, 14557281 was transferred into a new reaction tube and the pellet discarded. 50 ml of the cleared lysate were saved for determination of the loading control in SDS-PAGE and western blot. 1 mg of the antibody, was added to the lysate. The antibody/ lysate solution was then incubated for 2 h at 4uC with constant rotation. After 2 h, 30 ml of Protein G SepharoseTM 4 Fast Flow were added to the solution and the incubation was continued for 2 h at 4uC with constant rotation. The lysates were then centrifuged at 4uC for 5 minutes at 250 g. The supernatant was discarded and the pellet was washed twice with lysis buffer then twice with wash buffer and once with PBS. Between the washing steps the lysates were centrifuged at 4uC for 5 minutes at 250 g. The pellet was resuspended by flicking the tube gently and 50 ml of 26 sample buffer were added; the pellet was again resuspended by flicking and then boiled for 5 minutes at 95uC. After boiling, the samples were centrifuged for 3 minutes at 11,000 g and 20 ml of each sample supernatant were loaded onto a NuPAGEH 10% Bis-Tris Gel. SDSPAGE and western blot were performed as described above with primary antibody and Annexin-I Therapeutic Targets in Dowling-Meara Cell Lines loading control. Secondary antibodies: Goat anti-mouse IgG:HRP, Serotec, 1/1000 in