Tnt sp6 high yield expression system

Preparation of affinity Sepharose covalently coupled with each of the stereoisomers of CCG-1423 was performed based on previously reported methods [13 (link)]. MRTF-A protein was synthesized in vitro using the TNT SP6 High-Yield Expression System based on an optimized wheat germ extract (Promega) and was purified using anti-Flag M2 affinity gel. Mixtures of MRTF-A protein (300 ng), 0.005% bovine serum albumin, and the indicated CCG-1423 Sepharose or control Sepharose (bed volume 25 μl) in the pull-down (PD) buffer [13 (link)] (total 400 μl)] were incubated at 4°C for 2 h with rotation. After washing the respective Sepharose with the PD buffer and phosphate-buffered saline, the pull-downed MRTF-A protein was detected by IB.

MRTF-A- CRM1 interactions were performed as previously described14 (link)15 (link). The interactions between MRTF-A/B and NF−κB p65 were examined as follows. Flag-tagged MRTF-A/B and HA-tagged NF−κB p65 proteins were prepared using a TNT SP6 high-yield expression system based on an optimized wheat germ extract (Promega). The IP buffer mixtures (500 μl total volume) containing Flag-MRTF-A or Flag-MRTF-B proteins (15 μl) and HA-NF−κB p65 proteins (35 μl) were subjected to IP analyses as described previously12 (link). The IP buffer included 20 mM Tris–HCl, pH 7.5, 0.5% NP-40, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM β−glycerophosphate, and proteinase inhibitors (complete Mini; Roche Applied Science). Proteins in immunoprecipitates were detected by IB with the indicated antibodies. For these analyses, 3.3% of the input proteins and 22.2% of the IP proteins were subjected to IB, respectively. The IP/IB analyses using whole cell extracts were similarly performed. Quantification of the respective IP/IB signal intensities was performed using NIH ImageJ software.