Nf κb p65 c 20

RNA was isolated from the cells or mouse prostate tissues using Rneasy Kit (Qiagen) with the addition of Dnase I to minimize DNA contamination. Imprinting was performed using a FluPE assay as previously described [6] (link). For human IGF2, a single nucleotide polymorphism identified on IGF2 exon 7 (G/C) was used to identify individual alleles. IGF2 imprinting was examined on Exon 6 (A/G) in mouse prostate tissues. Differences were determined by calculating the ratio of their respective spectral intensities [repressed allele (Ai)/active allele (Aa)]. Quantitative PCR was performed using an iCycler (Bio-Rad) and SYBR Green PCR master mix (Applied Biosystems) to measure gene expression, primers are available on request. Western blot was performed to detect protein expression using antibodies for CTCF (Cat #07-729, Millipore), NF-κB p50 (4D1, Santa Cruz), NF-κB p65 (c-20, Santa Cruz), NF-κB p100/52 (18D10, cell signaling), IKKα/β (sc-7607, Santa Cruz) and IκBα (c-21, Santa Cruz) and α-Tubulin (DM1A, EMD).

Cells were collected after 72 h of treatment with the inhibitor or with DMSO as a control. Cell lysis (with RIPA) and protein quantification (Pierce BCA Protein Assay Kit, Thermo Scientific) were performed. Equal amounts of protein were loaded onto 12% SDS–PAGE gels. Gel electrophoresis and the subsequent Western blotting procedure were performed according to standard protocols. The Western blotting results were quantified using Image Lab software (version. 6.0.1., BIO-RAD, Hercules, CA, USA). The following antibodies were used: E-Cadherin (24E10), rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA); Slug (C19G7), rabbit monoclonal antibody (Cell Signaling); ß-actin (ab8227), rabbit polyclonal antibody (abcam, Cambridge, UK); and NF-κB p65 (C-20), rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA).

Total cell lysates were prepared by homogenization in modified RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors, as previously described (Castaldo et al., 2019 (link)). The membranes were incubated overnight with the following antibodies: Phospho-NF-κB p65 (Ser536) (93H1) (1:1000 dilution; Cell Signaling, Danvers, MA, United States #3033), NFκB p65 (C-20) (1:300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, United States #sc-372), IKKα/β and IKKγ (1:1000 dilution; Santa Cruz Biotechnology, #sc-7607 and #sc-8032, respectively), anti-ZNF224 (T3) (Cesaro et al., 2021b (link)) and anti-GAPDH (1:1000 dilution; Cell Signaling #2118). The blots were visualized using Clarity Western ECL Substrate Kit (Bio-Rad Laboratories) and immunoreactive bands were detected by autoradiography or by ChemiDoc XRS Image System (Bio-Rad Laboratories). Quantification of protein bands was obtained by densitometric analysis using the ImageJ software.

Cells were lysed in a 100 mM NaCl, Tris 50 mM pH8, NP40 1%, Glycerol 50% extraction buffer supplemented with a complete protease inhibitor cocktail (Roche, Meylan, France). After sonication, extracts were clarified by centrifugation, quantified by Bradford staining (Bio-Rad, Marnes-la-Coquette, France), denatured by heating (95 °C for 3 min) and separated by SDS-PAGE. Protein expression was examined using either the monoclonal anti-p21^CIP1/WAF1 clone SX118 (Agilent, Les Ulis, France), anti-QPRT (ab57125, Abcam), anti-apolipoprotein E clone E6D7 (Abcam), anti-p53 DO-7 (Agilent), anti-α-tubulin clone DM1A (Sigma), or the anti-β-actin clone AC-15 (Sigma) antibody, or the polyclonal anti-ZEB1 H102 (Santa Cruz Biotechnology, Heildelberg, Germany), anti-ZEB2 [22 (link)], anti-phospho-histone H3 (Ser10)-R (Santa Cruz Biotechnology), anti-phospho p53 (Ser15) (Cell signaling), anti-acetyl p53 (Lys373, Lys 382) (Millipore SAS, Molsheim, France), and the NF-κB p65 (C20) (Santa Cruz Biotechnology) antibody and horseradish-peroxidase-conjugated secondary antibodies (Dako). Western blots were normalized using the Image J software.