For stable cell line generation, inhibitor Y-27632 SCP2 cells were transfected with p21 shRNA and pools of stable cells were selected with 10 ng/ml puromycin. SUM159PT cells were serum starved for 24 hrs in the absence of insulin and hydrocortisone before TGFb1 stimulation. Western blot analysis and immunoprecipitation Cells were lysed in cold extraction buffer containing protease inhi bitors. The lysates were then centrifuged at 14,000 rpm for 15 minutes at 4 C. Protein content was measured using BCA protein assay kit. Equal protein was analyzed by Western blot using mouse anti p21, mouse anti c myc, mouse anti p15, rabbit anti Smad2/3, phospho cofilin and cofilin antibodies, and fol lowed by secondary antibodies goat anti mouse or rabbit. Immunoprecipitations were performed overnight at 4 C using antibodies against p300/CBP, p/CAF and p21.
Protein G Sepharose was added for 1 hr at 4 C, and washed four times with cold lysis buffer. The immunocomplexes were boiled with 2�� sodium dodecyl sulfate Laemmli sample buffer for five minutes and subjected to immunoblotting. Histone proteins extraction Total histone proteins were extracted as previously described. Briefly, 80% confluent of SCP2 cells from a 100 mm tissue culture plate were serum starved for 24 hrs and stimulated with or without 5 ng/ml TGFb or 1 ��M trichostatin A. SCP2 cells were harvested and resuspended in cold hypotonic lysis buffer containing 10 mM Tris HCl, pH 8. 0, 1 mM KCl, 1. 5 mM MgCl2, 1 mM DTT, protease inhibitors, 1 ��M TSA and 10 mM sodium butyrate. Cell lysates were rotated at 4 C for 30 minutes and then centrifuged at 10,000 g, 4 C, for 10 minutes.
The supernatants were discarded and nuclei pellets were resuspended in 400 ��l of 0. 4 N H2SO4 and incubated overnight on a rotator at 4 C. Samples were centrifuged at 16,000 g for 10 minutes and supernatants containing histones were transferred into a fresh tube. A total of 132 ��l trichloroacetic acid was added drop by drop to the histone solution, inverted several times and then incubated on ice for 30 minutes. The histone precipitates were centrifuged at 16,000 g for 10 minutes and pellets were washed twice with ice cold acetone and the histone pellets were air dried for 20 minutes. Total histone pro teins were subjected to Western blot analysis using an acetylated lysine antibody.
DNA affinity precipitation assay SCP2 cells transiently transfected with the indicated siR NAs were stimulated with TGFb for 30 minutes. Cell lysate were extracted in cold lysis buffer containing 10 mM Tris HCl, pH 7. 4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P 40, 30 mM sodium pyrophosphate, 1 mM sodium Carfilzomib orthovanadate and protease inhibitors as described above. A total of 5 ��g Poly competitor was incubated with 1 mg of total cell lysate for 30 minutes at 4 C. A total of 500 pmol of double stranded oligonucleotides was added and incu bated with cell lysates for two hours at 4 C.
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