The streptavidin agarose beads containing biotinylated oligonucleotides and protein complex were boiled with 2�� SDS Laemmli sample buffer for five min utes and subjected to immunoblotting. The sequences of biotin labeled double strand oligonucleotides were previously described. For Smad binding ele ment oligonucleotide Luciferase assays SCP2 selleck chemicals llc cells were transiently co transfected with 50 nM Scr siRNA, 50 nM p21 siRNA or 0. 5 ��g flag tagged p21 cDNA in combination with 0. 3 ��g SBE reporter construct and 0. 1 ��g pCMV b gal. Transfected cells were then stimulated with or without 5 ng/ml TGFb for 16 hrs. Luciferase activity of CAGA12 luc was measured and nor malized to b galactosidase activity. Real Time PCR Total RNA was extracted using TRIzol reagents.

Reverse transcription of total RNA using random primers was carried out using M MLV reverse transcrip tase as per the manufacturers instructions. Real time PCRs were carried out using SsoFast Eva Green Supermix in a Rotor Gene 6000 PCR detection system. Thiazolyl blue tetrazolium bromide assay A total of 100 ��l of cell suspension was stimulated or not in the presence or absence of 5 ng/ml TGFb and cultured in 96 well plates for two days. After two days, 25 ��l 5 mg/ml MTT solution was added to each well and incubated for two hours. A total of 200 ��l of dimethyl sulfoxide was added to each well and mixed well. The absorbance at 570 nm was mea sured on a plate reader. Cell cycle analysis SCP2 cells were stimulated with TGFb for 0, 2, 6 and 24 hrs. Cells were then fixed with 70% ethanol over night, treated with 20 ��g/ml RNase, and stained with 0.

5 mg/ml propidium iodide. DNA content was determined using a FACScan flow cytometry analyzer. Kinetic cell migration assay Cells were transfected with different siRNAs and plated in Essen ImageLock 96 well plates at 50,000 cells per well. The use of ImageLock 96 well plates ensures that images/videos of the wound are automatically taken at the exact same loca tion by the IncuCyte Brefeldin_A software. Cells were then serum starved for six hours and confluent cell layers were scratched using the Essen Wound maker to generate approximately 800 ��m width wounds. After wounding, cells were washed two times with PBS and sti mulated in the presence or the absence of 5 ng/ml of TGFb. ImageLock 96 well plates were then placed into IncuCyte and imaged every hour for 24 hrs. The data were analyzed by three integrated metrics wound width, wound confluence or relative wound density automatically measured by the IncuCyte software. Matrigel invasion assay For the Transwell assays, 30 ��l of growth factor reduced Matrigel was coated onto each insert of 24 Tranwell inva sion plate and incu bated for two hours in the cell culture incubator.