DCs (1 × 107 cells) were stimulated with LPS (0.1 μg/mL), with or without Cl-amidine (200 μM), for 1 h, and then nuclear proteins were isolated using the Nuclear Extraction Kit, following the manufacturer’s instructions (Abcam). To determine AP-1 family member activity, ten micrograms of total nuclear protein used with the AP-1 Transcription Factor Assay Kit, according to the manufacturer’s instructions (Abcam).
Ap 1 transcription factor assay kit
Manufactured by Abcam
Sourced in United States
The AP-1 Transcription Factor Assay Kit is a sensitive and specific tool for the detection and quantification of active AP-1 transcription factor in cellular extracts. The kit provides a simple and efficient method for measuring AP-1 DNA-binding activity.
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4 protocols using ap 1 transcription factor assay kit
1
AP-1 Activation in LPS-Stimulated DCs
2
Profiling AP-1 Transcription Factors
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Wild-type BMDMs were stimulated with IL4+IL13+TNF for 15 min (Fig S7F ) or with IL4+IL13 for 12 h before TNF was added for further 150 min (Fig S7G ). Nuclear extracts were prepared with the NE-PER Nuclear and Cytoplasmic kit (78833; Thermo Fisher Scientific). AP-1 transcription factor assay kit (ab207196; Abcam) was used according to the manufacturer’s instructions.
3
Quantifying AP-1 Activity in Glioma
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Two IL-13Rα2 positive (U251 and A172) and one IL-13Rα2 negative glioma cell line (T98G) were grown in complete medium and stimulated with 20 ng/ml IL-13 for 30 min when the tumor cells were in log phase. Nuclear proteins were isolated using the nuclear extraction kit following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). 10 µg of total nuclear protein was used to determine AP-1 family member activity using the AP-1 transcription factor assay kit as recommended by the manufacturer’s instructions (Abcam, Cambridge, MA). The assay was performed in quadruplicate and results were expressed in arbitrary units after measuring optical density at 450 nm within 5 min with a reference wavelength absorbance at 665 nm.
4
Quantifying AP-1 Transcription Factor Activity
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Nuclear fractions were obtained from MDCK cells (~90% confluent) using the Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce, Rockford, IL, USA) following the manufacturer's instructions. The protein content of nuclear fractions was determined by using the BCA kit (Pierce). AP-1 activity was measured using the commercial enzymelinked immunosorbent assay (ELISA)-based AP-1 Transcription Factor Assay Kit (Abcam, Cambridge, MA, USA) as reported [26, (link)27] (link). DNA specific binding activity of AP-1 was measured using nuclear extracts against the AP-1 binding site 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE) consensus sequence 5´-TGAGTCA-3′ fixed in a 96-well plate. Specific activity was determined for the Fos family members cFos, FOS-B, and Fra-1, and the Jun family members phosphorylated cJun (p-c-Jun), Jun-N, and Jun-D. Specific primary antibodies and HRP-conjugated secondary antibodies included in the commercial ELISA kit were used. Absorbance was detected at 450 nm.
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