Ab70892

For total protein extraction, hPSCs were lysed with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors cocktail 2 and 3 (Sigma-Aldrich). For histone extraction we used the Histone extraction protocol for western blot from Abcam (Cambridge; UK) web page. Cell lysates were separated by molecular weight using SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Protein was detected using the Odyssey Infrared Imaging System (Li-cor Biosciences; Lincoln, NE, USA). To detect KDM5A and NUP98-KDM5A was used the α-KDM5A antibody (ab70892, Abcam). Also used α-NUP98 (ab50610, Abcam), α-HIF1A (610959, BD Bioscience; San Jose, CA, USA), α-γ-H2AX (#9718, Cell signalling; Danvers, MA, USA). An α-β-Actin (A5441, Sigma-Aldrich) was used as a loading control for total protein extractions and α-H3 (ab1791, Abcam) was used as a loading control in histone extractions. Western blotting was carried out using standard procedures. Quantification of band intensity and normalization was carried out using ImageJ (NHI, Bethesda, Maryland, USA, https://imagej.nih.gov/ij).

One milligram of nuclear extracts was diluted in dilution buffer (50 mM Tris, pH 7.5, 0.3% NP-40, protease inhibitor, 1 mM DTT) to attain a final concentration of 150 mM NaCl. Nuclear extracts were pre-cleared for 3 h at 4°C by incubation with normal IgG (goat sc-2028 and rabbit sc-2027; Santa Cruz) and protein G Dynabeads (Life Technologies), blocked with 0.2 mg ml⁻¹ BSA (Pierce) and 0.4 mg ml⁻¹ sonicated salmon sperm DNA (Life Technologies). Immunoprecipitations were performed overnight at 4°C by incubation of the pre-cleared nuclear extracts with primary antibodies (for JARID1A, ab 70892; Abcam) and blocked protein G Dynabeads in dilution buffer (50 mM Tris, pH 7.5, 0.3% NP-40, protease inhibitor, 1 mM DTT). Beads were washed five times with 4× bed volume of wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.3% NP-40, protease inhibitor, 1 mM DTT) and bound material was eluted in 1× Laemmli buffer by boiling at 95°C for 10 min. Fractions were analysed by immunoblotting as described above.

The indicated plasmids (Flag-Fbxo22, HA-KDM5A, and V5-Ubiquitin) were transfected into HEK293T cells. Prior to collection, cells were treated as previously described (Zhang et al. 2019 (link)). The obtained lysates were assayed and immunoprecipitated with protein A/G agarose (Sigma) pre-conjugated with the indicated antibody anti-HA (1:50, #3724, Cell Signaling). In addition, to prevent detection of ubiquitination of the E3 ligase itself and proteins associated with KDM5A, ubiquitination assays were also performed under denaturing conditions. Transfected cells were lysed in lysis buffer and incubated with SDS-PAGE sample buffer at 100 °C for 8 min, followed by incubation with HA antibody for IP and Immunoblotting. Antibodies used for immunoblotting were anti-flag (1:50, F3165, Sigma), anti-HA (1:50, #3724, Cell Signaling), and anti-V5 (1:50, 13,202, Cell Signaling).
Additionally, the ubiquitination level of KDM5A in TNBC cell lines was also examined. The obtained lysates were immunoprecipitated with protein A/G agarose (Sigma) pre-conjugated with the antibody of anti-KDM5A (1:100, ab70892, Abcam) or anti-IgG (1:50, #3900, Cell Signaling). The expression of the relevant proteins was then detected by immunoblotting using the antibodies of mouse anti-KDM5A (1:1000, ab78322, Abcam) and anti-ubiquitin (1:1000, 04–263, Millipore).