Nuclear extraction kit

HUVECs were treated with proHp CM (final proHp: 0.2 μg/ml), and incubated for 14 h. Nuclear proteins from the cells were extracted with the Nuclear Extraction Kit (Signosis, Santa Clara, CA, USA), and transcription factor activation was detected using the Transcription Factor Activation Array І (Signosis), according to the manufacturer’s instructions. Briefly, the nuclear extracts were incubated with the probe mix for 30 min at room temperature, and then the transcription factor/probe complexes were bound to a spin column. The bound probes were separated from the complexes with elution buffer and hybridized to plates precoated with DNA sequences that are complementary to the specific probes. The captured probes were detected with streptavidin-HRP. The luminescence was measured on a Victor3 luminometer (PerkinElmer, Boston, MA, USA) and expressed as relative light units (RLUs).

We used the transcription factor activation profiling plate array (synthesized by Signosis, Sunnyvale, CA) to analyze the activity of different transcription factors in HT-29 and WiDr colon cancer cells treated with NCTD for 24 h according to the manufacturer’s instructions. First, nuclear proteins were extracted from HT-29 and WiDr cells using a Nuclear Extraction Kit (Signosis, Sunnyvale, CA). Then, 15 μg of nuclear protein extract was mixed with biotin-labeled probes based on the consensus sequences of transcription factor DNA binding sites. Thus, transcription factor/probe complexes were formed. Next, we separated the transcription factor-DNA complexes from free probes, transferred the complexes to a PCR tube, and denatured the eluted probes. After separation of the bound probes from the complexes, they were hybridized with sequences complementary to the probes in the hybridization plate. Finally, the captured DNA probes were detected with streptavidin-HRP, and the signal intensity was measured based on relative light units (RLUs) using a microplate luminometer.

Cell lysate after treatment was prepared using Signosis nuclear extraction kit (SK-0001). Equal amount of nuclear lysates from treated or untreated cells were mixed with biotinylated STAT3 or NFκB probes from Signosis EMSA assay kit. After 30 mins incubation at 22° C, samples Were run on a 6.6% polyacrylamide gel using 0.5x Tris Borate buffer (TBE). Samples with bound probes Were transferred onto a nitrocellulose membrane. The membrane subjected to UV irradiation On a Biorad GS gene linker with 125 milli Joules for 2 minutes. Immobilized probes were incubated With Steptavidin-HRP and were detected suing chemiluminiscence substrate. Image was obtained on a Licor Odyssey FC machine.