Protein g agarose beads

MDA-MB-231, MDA-MB-468, and Hs578T cells were harvested after 4 h treatment with DPEITC or DMSO (as a control), and lysates were processed for immunoprecipitation, as described previously [20 (link),21 (link)]. Briefly, 150 μg of the lysates were precleared at 4 °C for 30 min with protein G-agarose beads (#11719416001, Millipore Sigma, Rockville, MD, USA). The precleared lysates were then gently tumbled at 4 °C for 2 h with a mutant p53 conformation-specific mouse PAB240 antibody (#OP29, Calbiochem, Millipore Sigma, Rockville, MD, USA), followed by the addition of protein G-agarose beads and additional incubation for 2 h at 4 °C. The beads were washed three times with lysis buffer, and immunoprecipitated proteins eluted in Laemmli buffer were analyzed by Western blotting with a rabbit polyclonal FL393 (sc-6243, Santa Cruz Biotechnology, Dallas, TX, USA) as a primary antibody. For the secondary antibody, peroxidase-labeled mouse anti-rabbit IgG (1:5000, sc-2357, Santa Cruz Biotechnology, Dallas, TX, USA) was used. The blot was developed using the ECL Prime Western Blot Detection Kit according to the manufacturer’s protocol. As an input control, the blot was probed with p53 (DO-1) and then re-probed with an anti-GAPDH antibody.

Nuclei isolated from HEK293T cells were lysed in Co-IP buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 0.5% NP-40 and 2% glycerol) supplemented with protease inhibitors (Complete EDTA-free, Roche Applied Science). NaCl and glycerol concentrations were adjusted to 150 mM and 1%, respectively. Lysates were then pre-cleared with Protein G-agarose beads or Protein A-agarose beads (Millipore) on a rotating shaker at 4°C for 2.5 hrs. Pre-cleared lysates were incubated with specific antibodies or relative normal control IgGs (Santa Cruz Biotechnology) on a rotating shaker overnight at 4°C. Agarose beads, previously saturated with BSA (1 μg/μl) overnight, were added to the lysate/antibody solutions and incubated for 3 hrs on a rotating shaker at 4°C. Subsequently, beads were washed 10 times in Co-IP wash buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40 and 1% glycerol), proteins were eluted boiling in 2X Laemmli buffer and then analysed by Western blotting.

Metabolic labelling assays were performed as previously described [5 (link)] with the following modifications. Cells were serum starved in DMEM without methionine and cysteine (ThermoFisher Scientific, Cat. 21013024) for 45 min, and pulse-labelled in 100 μCi/mL EasyTag™ Express Protein Labeling Mix, [35S] (Perkin Elmer, Cat. NEG772) for 15 min (WT CFTR) or 1 hour (ΔF508-CFTR). Cells were rinsed with cold PBS containing 1mM MgCl₂ and 0.1mM CaCl₂ and chased in DMEM supplemented with 10% FBS, 2 mM glutamine, 2 mM methionine and 2 mM cysteine. Cells were harvested at 0, 1, 2, or 3 h in 500 μL RIPA buffer. Soluble material was separated by centrifugation at 20,000 x g for 5 min at 4°C. Protein concentrations were normalized using the BCA Protein Assay Kit, and pulse-labelled CFTR was isolated by immunoprecipitation with a mixture of CFTR monoclonal antibodies (Millipore, MAB3480 (1.5 μg) and MAB3484 (1.5 μg) per sample) and 50 μL protein G agarose beads (Millipore, Cat: 16–266) for 2.5 hours at 4°C. Beads were washed 3 times in RIPA buffer and proteins were eluted in Laemmli loading buffer. CFTR bands were separated by 6% SDS-PAGE and radioactive bands were detected using a Typhoon scanner (GE Healthcare), then quantified using ImageJ 1.46r software.

SMMC-7721 cells were resuspended in 1 mL of RIPA lysis buffer (Beyotime) and pre-cleared with protein G agarose beads (Millipore, Billerica, MA, USA) for 1 hour. Supernatants were incubated with ARID2 or E2F1 antibodies overnight at 4°C, followed by a two-hour incubation with protein G agarose beads. Immune complexes were resolved by 10–15% SDS/PAGE and transferred to PVDF membranes, then subjected to immunoblot analysis with the indicated antibodies.