P53 doi

Western blotting experiments and/or IF and IF combined to FISH were performed using the following primary antibodies: PSPC1 (Sigma-Aldrich, SAB4200503), PSF (Sigma-Aldrich, P2860), FBXO11 (Novus Biologicals, NB100-59826), TCF7L2 (Cell Signaling Technology, 2565), CPSF1 (Santa Cruz Biotechnology, sc-166281), CPSF2 (Santa Cruz Biotechnology, sc-165983), GAPDH (Abcam, ab9485), NONO (Bethyl Laboratories, A300-587A), TDP-43 (ProteinTech, 12892-1 ap), CPSF3L or INTS11 (Sigma-Aldrich, A107128), INTS1 (Millipore), INIP [C9orf80 (E-12), Santa Cruz Biotechnology, SC-137357], INTS3 (Bethyl Laboratories, A300-427A), INTS6 (Bethyl Laboratories, A301-658A), INTS8 (Bethyl Laboratories, A300-269A), p53-DOI (Santa Cruz Biotechnology, sc-126), p21 (Santa Cruz Biotechnology, sc-6246), phospho-γH2AX (Cell Signaling Technology, 2577), Laminin A + C (Abcam, ab108922), vinculin (Sigma-Aldrich, V9131), GFP (Clontech, 632375), and H3 (Abcam, ab1791).

Cells were lysed in 200–500 μL of RIPA buffer (Thermo Fisher Scientific 89901) containing protein inhibitors cOmplete, Mini, EDTA-free protease inhibitor cocktail (Millipore Sigma 11836170001). Subsequently, samples were sonicated twice for 5 sec (power set to 50%) while samples were on ice. Cleared supernatant from lysates were recovered by centrifugation and the protein was quantified using Pierce BCA protein assay kit (Thermo Scientific 23225) according to the manufacture's protocol. Protein (15 or 20 μg) was loaded into 6%, 10%, or 12% SDS-polyacrylamide gels and, subsequently, transferred onto a PVDF membrane in a semi-dry apparatus according to standard Western Blot protocols. Membranes were blocked and later they were incubated with primary antibodies overnight. We used GAPDH antibody (1:6000 dilution; Cell Signaling 5174S) or β-tubulin (1:1000 dilution; Cell Signaling 2128S) as a normalizer for protein expression. The other primary antibodies used are the following: FLAG (1:1000 dilution; Sigma F1804), p53 (DO-I) (1:1000 dilution; Santa Cruz Biotechnology sc-126), CD44 (1:1000; dilution Cell Signaling, Catalog no. 3570S), mouse IgG (Santa Cruz Biotechnology sc-2025) and ZMAT3 (1:250 dilution), that was generated from Abgent. Membranes were developed using ECL Prime Western blotting detection reagent (Fisher Scientific RPN2232).