Nf κb p65 rela

Total proteins from the cells or tissues were extracted with RIPA lysis buffer containing proteinase inhibitor. The protein concentrations were measured by the BCA reagent kit (Beyotime). The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, which were blocked in 5% non-fat milk, and then incubated with the primary antibodies against ARID1A (Bethyl Laboratories), NF-κB p65 (RELA) (Santa Cruz), E-cadherin (Santa Cruz), CagA (Santa Cruz), p21 (Proteintech) and β-actin (Abcam) at 4 °C overnight. The membranes were then washed in tris buffered saline tween and incubated with anti-mouse or rabbit horseradish peroxidase-conjugated secondary antibodies and developed with the enhanced chemiluminescence method (ECL, Millpore). β-actin served as a loading control.

3 × 10⁷ cells were lysed in 1 ml hypotonic buffer (10 mM HEPES, pH 7.9; 1.5 mM MgCl₂; 10 mM KCL) containing 0.15% NP-40 at 4°C for 10 min. The nuclear proteins were extracted from the nuclear fraction by suspending the nuclei in 100 μl extraction buffer (300 mM KCl; 1.5 mM MgCl₂; 20 mM HEPES, pH 7.9; 0.2 mM EDTA; 25% Glycerin) at 4°C for 20 min with constant agitation. A CSL site spanning oligonucleotide (5′-CAGCCCTGTGGGAACTTGCTG-3′) was annealed with the reverse complementary strand and served as probe. EMSA for the detection of NOTCH/CSL complexes were performed essentially as described (Hubmann et al., 2002 (link)).
The N1^IC (bTAN 20) and N2^IC (C651.6DbHN) antibodies used for supershift/interference and western blot assays were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Department of Biological Science, Iowa City, IA, United States). The NFκB p65 (RelA) and ACTB Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Western blotting was performed according to standard protocols.