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. The 
cells were covered with lysis buffer, scraped off with a rubber policeman and transferred into microcentrifuge tubes. The cells were lysed by pipetting three times through a 22G syringe and then incubated at 95uC for five minutes. The cell lysates were stored at 20uC. Western Blot Protein levels were determined by SDS-PAGE and western blot analysis on NuPAGEH 10% Bis-Tris Gels using 20 ml cell lysate or concentrated cell culture MedChemExpress SCH58261 supernatant of each determined cell line. Annexin-I was used as a loading control for cell lysates. As a negative control for the western blot, one randomly chosen extra sample was applied; all gels included lanes with 4 ml of size marker. The electrophoresis run was performed at 100 Volts for about 2 h in 16 NuPAGEH buffer. The size-separated proteins on the polyacrylamide gel were then electrophoretically transferred to a nitrocellulose membrane. Blotting was done at 4uC at 250 mA for 1 h in transfer buffer. The membrane was then incubated in blocking solution for 1 h at room temperature to saturate unspecific antibody-binding sites. The blocking reagent was then discarded and the primary antibody was applied. Primary antibodies were incubated at 4uC overnight. No primary antibody was applied to the negative control. The next day, the primary antibody solution was washed away with blocking solution three times for ten minutes at room Isolation of Proteins from Cell Culture Supernatant For western blot analysis of kallikrein-related peptidases, cells were incubated for 48 h in EpilifeH medium without FCS. The supernatant was collected and filtered through a cell strainer to remove dead cells and cell debris. Complete mini protease inhibitor was added to the supernatant and the solution was concentrated by centrifugation through a centrifugal filter for 20 minutes at 4uC and 6800 g. The concentrate was mixed with 46 sample buffer and subject to SDS-PAGE and western blotting as described below. Collection of Blister Fluid Patients of various EBS subtypes visited the Dermatology Department of Paracelsus Medical University and the EB house Austria for routine check up and wound care. 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 20719936 two layers of transparent 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 11408530 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 a