The absorbance value was measured at 570 nm using a multiwell spectrophotometer

tibodies for 1 hour. After washing, the cells were incubated with the appropriate fluorescence-conjugated secondary antibodies for 1 hour. Slides only incubated with the secondary antibodies were used as controls for non-specific signal. Cells were washed in 0.2% BSA-PBS, briefly rinsed in 2 mM Hoechst 33258 reagent to stain nuclei, and mounted with ProLongH antifade. Stained cells were photographed with 17016504 a Nikon Eclipse TE 2000-U confocal microscope. Western blot analyses Western blot analyses were essentially performed as described previously. Cells were lysed in ice-cold lysis buffer and protein 18673174 concentrations were determined. Protein samples were separated by SDS-PAGE, blotted onto PVDF membranes, and incubated with the primary antibody. Following incubation with horseradish peroxidase-conjugated secondary antibody, the bands were visualized with a luminol-based detection system with p-iodophenol enhancement. Anti-tubulin antibody was used to confirm equal loading of protein in each lane. Some membranes were re-probed with several antibodies using a stripping solution and following the manufacturer’s instructions. RT-PCR analysis Total RNA was isolated using Nucleospin RNAII, according to the manufacturer’s instructions. Single-strand cDNA was generated from 2 mg of total RNA using poly-dT as primer with M-MLV reverse transcriptase. For RTPCR, 1 ml of cDNA was used in a standard 50-ml PCR mixture with 400 nM of each primer and 2 U of FastStart Taq DNA polymerase. The PCR products were separated by electrophoresis on a 1% agarose gel and visualized by SybrSafe staining. Quantitative RT-PCR was performed in triplicate. Each 20 ml reaction contained 1 ml of cDNA, 400 nM of each primer, and 1x iQ SybrGreen Supermix. Standard curves were run for each transcript to ensure exponential amplification and to rule out non-specific amplification. Gene expression was normalized to RPS13 expression. The reactions were run on an iQ5 Real-time PCR detection system. The specific primers used for PCR are described in Retroviral transduction 293T cells were plated on a 10 cm dish, incubated overnight, and then co-transfected according to the calcium phosphate precipitation method with 10 mg of pCL-Eco plasmid containing the gag, pol and env viral proteins and 10 mg of a pBabe-puro retroviral vector containing the human endoglin gene. After 48 hr, the virus-containing medium was filtered and supplemented with 4 mg/ml polybrene . Viral supernatants were Plasmids, transfection, and luciferase reporter assays The expression plasmids for human endoglin have been described previously. ON-TARGETplus SMARTpool siRNA against ENG and control siRNA were obtained from Dharmacon. The TGFb-responsive vectors used as reporters were the ALK5-Smad3-specific 12-Luc , the specific Smad2-responsive Fast/pAre-Luc, and p2-Luc, which contains ALK1-specific response elements. In the luciferase Endoglin Regulates Dermal Fibroblast through Akt assays, the expression plasmid pRL-TK vector containing the Renilla luciferase gene served as an internal control to correct transfection efficiency. Cells were transfected using Lipofectamine 2000 for 5 hours, according to the manufacturer’s instructions. Luciferase and Renilla activities were measured using a dual-reporter assay kit. 5 Endoglin Regulates Dermal Fibroblast through Akt Proliferation assays For MedChemExpress Nigericin (sodium salt) crystal-violet assays, 5000 cells were seeded on a 24-well plate and incubated in 10% FCS medium with or without additional agonis