Journal: The international journal of biochemistry & cell biology
Article Title: N-Terminal Truncation of Stat5a/b Circumvents PIAS3-Mediated Transcriptional Inhibition of Stat5 in Prostate Cancer Cells
Figure Lengend Snippet: Short form STAT5 occurs naturally in prostate cancer cells A , Electromobility shift assay of nuclear extract of DU145 cells prepared from flash frozen cell pellets in the absence (lane 1 and 2) or presence (lane 3 and 4) of protease inhibitors (phenylmethylsulfonylfluoride, aprotinin, leupeptin and pepstatin-A). The short form Stat5a/b (arrow) was detected in DU145 cells in the presence or in the absence of protease inhibitors, and the short form Stat5a/b was super-shifted by anti-Stat5a and anti-Stat5b antibodies. B , The short form Stat5a/b is not generated in vitro after disruption of the cell membranes. DU145 cell pellets were flash frozen in liquid nitrogen and directly lysed in boiling SDS loading buffer (lane 1), or the cell pellets were lysed in cell lysis buffer in the presence (lane 2) or in the absence of proteases inhibitors (lane 3) and boiled in SDS loading buffer and analyzed by Western blotting using anti-C-terminus Stat5a/b mAb.
Article Snippet: Cells were lysed and fresh human prostate cancer specimens were homogenized in lysis buffer [10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulphonylfluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin], and the protein concentrations of the whole cell lysates were determined by the Bradford method (BioRad Laboratories Inc., Hercules, CA).
Techniques: Electro Mobility Shift Assay, Generated, In Vitro, Lysis, Western Blot