Ab133112

Nuclear extracts were isolated from heart and brain tissues collected at R30 using a commercially available nuclear extract kit (EMD #2900, Billerica, MA). The DNA binding activity of NF-κB (p50/p65) in the nuclear fraction was determined using an ELISA-based nonradioactive NF-κB p50/p65 transcription factor assay kit according to the manufacturer's protocol (Abcam #ab133112, Cambridge, MA).

ELISA-based transcription factor assay kit (ab133112, Abcam) was used to determine the EGCG effect on p65-NF-κB activity in human pancreatic cell lines (MIAPaCa-2 and SU.86.86). The nuclear extract was extracted by lysing the treated cells with EGCG (10–100μM for 24 h) using hypotonic HEPES lysis buffer (pH 7.4) provided with a nuclear extraction kit (ab113474, Abcam). As recommended by the manufacturer, the supernatant was taken and utilized to measure intracellular p65-NF-κB by ELISA. In brief, the p65-NF-κB response element with a specific double-stranded DNA (dsDNA) sequence was immobilized onto the bottom of wells in a 96-well plate. The p65-NF-κB contained in a nuclear extract bind specifically to the p65-NF-κB response elements. The p65-NF-κB was detected by adding a specific primary antibody directed against p65-NF-κB. A secondary antibody that was HRP-conjugated was added, and a spectrophotometer was used to measure the absorbance at 492 nm.

Inflammation markers including TNF-α (ab46070), IL-1β (ab100768), IL-6 (ab100772) and IL-10 (ab133112) ELISA kits were obtained from Abcam Co., Eugene, OR, USA. As for the apoptotic signaling markers, cleaved caspase-3 (KHO1091) was purchased from Thermo Fisher Scientific Inc., Waltham, MA, USA, whereas caspase-9 (LS-F4141) was acquired from Biocompare, San Francisco, CA, USA. These markers were measured according to the manufacturer’s instructions using a microplate reader SpectraMax i3X (Molecular devices, San Jose, CA, USA).

For the measurement of NF-κB activity, the nuclear fraction was first extracted by using an NE-PER nuclear extraction kit (Thermo Fisher Scientific, Waltham, MA, United States) and then subjected to an NF-κB activity assay (Abcam, ab133112, Cambridge, MA, United States).