Bl21 de3 star plyss cells

GST‐FOXM1 fusion constructs were expressed in BL21‐Star™ (DE3) pLysS cells (Thermo Fisher Scientific), and crude bacterial lysates were prepared by sonication in cold lysis buffer (50 mm Tris/HCl pH 8.0, 100 mm NaCl, 1 mm EDTA, pH 8.0, 2% NP‐40, 1 mm DTT, 1 mm PMSF). To test the interaction between FOXM1 and PMLIV, GST‐fusion proteins were freshly purified by glutathione‐Sepharose beads (GE Healthcare, Chicago, IL, USA), washed two times with lysis buffer and one time with GST‐Wash buffer (300 mm KCl, 20 mm HEPES pH 7.9, 0.1% NP40, 5 mm MgCl₂), and resuspended in 200 μL GST‐interaction buffer (150 mm KCl, 20 mm HEPES pH 7.9, 0.1% NP40, 5 mm MgCl₂) and mixed with 200 μg of HEK‐293T cell lysate overexpressing (OE) mRED‐PMLIV. The binding reaction was incubated for 3 h at 4 °C. Beads were washed three times with GST‐Wash buffer (600 mm KCl, 20 mm HEPES pH 7.9, 0.1% NP40, 5 mm MgCl₂) and resuspended in SDS sample buffer. Samples were subjected to SDS/PAGE and analyzed by immunoblotting.

The GST-tagged PTK6 fusions and PTEN proteins were expressed in BL21 Star (DE3)pLysS cells (Life Technologies, Carlsbad, CA). Protein expression was induced with the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and purification was carried out as previously described^{63 (link)}. For GST pull-down experiments, 5 µg of murine PTK6 GST-fusion proteins (GST, SH3, SH2, SH3/SH2, SH1, and full-length) were incubated with 30 µl glutathione-Sepharose 4B beads (50% slurry) for 30 min; 500 of µg of DU145 total cell lysate was then added and incubated overnight at 4 °C. Beads were washed 4 times in wash buffer (1% Triton X-100, 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM Na-pyrophosphate), resuspended in 2× SDS loading buffer then boiled for 5 min and resolved by SDS–PAGE.