Characterizing KDM5A Ubiquitination in TNBC

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).