Materials and methods Materials and solutions The recombinant human TGF B1 was obtained from Peprotech. The following reagents were purchased from e-book Cell Signaling Technology Com pany rabbit anti rat threoninetyrosine diphosphorylated ERK12 and total ERK12 antibodies, MAPK inhibitor, Inhibitors,Modulators,Libraries serine phosphorylated AKT, AKT and PI3K inhibitor. Rabbit anti rat caveolin 1 antibody was purchased from Abcam Company. RXM and B cyclodextrin were from Sigma Chemical Company. TGF B1 and RXM were taken pure water and dimethyl sulfoxide as their stock solutions, respectively. The final DMSO concentration of less than 0. 2% had no significant effect on cells growth. Animals and experimental protocol Male Sprague Dawley rats were purchased from SLAC Laboratory Animal Co, Ltd and divided into control and asthma groups.

The animals had no access to solid food but gained free access to water 12 hours before the experi ments. Inhibitors,Modulators,Libraries The experimental protocol was approved by the Committee of Animal Care in Wenzhou Medical Univer sity. All animals were handled in accordance with the Guideline for the Care and Use of Laboratory Animals. The ovalbumin model was constructed as previously reported firstly, the rats were sensitized with intraperitoneal injection of 10% OVA mixed with 10% alumin hydroxide solution, and then from day 15, the rats were challenged for 30 min by an aerosol of 1% OVA in normal saline, twice a week for 8 weeks. In control group, OVA was replaced by saline during sensitization and challenge. After the last challenge, 10% chloral hy drate was used for all animals euthanasia.

Preparation of ASMCS ASMCs Inhibitors,Modulators,Libraries were isolated from male SD rats. Briefly, rat bron chi were isolated under sterile conditions. After the con nective tissue and epithelia were removed carefully, the smooth muscle strips were Inhibitors,Modulators,Libraries cut into small pieces and digested in phosphate buffer saline, containing 0. 2% type I collagenase, at 37 C in a 5% CO2 and 95% air atmosphere for 20 min. The cells were dis persed and centrifuged at 1000 g for 5 min, and then the pellets were collected and resuspended in RPMI 1640, containing 10% FBS and 1% penicillin streptomycin. Fi nally, the cultures were maintained in a humidified atmosphere with 5% CO2 at 37 C. In each group, total six rats were utilized and all experiments were repeated in cells from the six different rats.

For all experiments, cells were plated into tissue culture flasks or Petri dishes and grown to 80% confluence. Furthermore, cells were serum deprived for 24 hours in RPMI 1640 prior to the addition of TGF B1. Stimuli and inhibitor ASMCs were incubated in the low serum Inhibitors,Modulators,Libraries medium with or without TGF B1 for 48 h. The inhibitors, including wortannin and PD98059, and B CD which could destroy caveolae were used. Finally, 0. 4 uM wortannin, 10 uM PD98059, and 10 mM B CD were added to ASMCs 1 h prior to each treatment according neither to manufacturers instructions.