Immunoprecipitation and ubiquitination assays were performed in accordance with previously published literature [27 (link),28 (link)]. For immunoprecipitation, the cells were rinsed twice with 1 × PBS, lysed with Cell Signaling Technology (CST) lysis buffer (CST9803, CST, Beverly, MA, USA) containing protease inhibitor (Sigma) at 4°C and centrifuged for 10 min to remove the pellets. Next, 1/10 of the cell lysis buffer was taken as the Input and the remaining part was cultured with anti-KDM3A (dilution ratio of 1:30, ab243641, Abcam) and protein A/G-Sepharose overnight at 4°C. After 5 washes with CST lysis buffer, the precipitated proteins were eluted with SDS-loading buffer and then incubated with anti-USP22 (dilution ratio of 1: 2000, ab195289, Abcam) for Western blot analysis.
Ubiquitination determination was carried out as the transfected cells were lysed using RIPA buffer containing 0.1% SDS to a final concentration of 0.2% SDS, and then subjected to identical operations as the immunoprecipitation detection. In addition, Western blot analysis was conducted using an anti-Ub antibody (dilution ratio of 1:1000, ab134953, Abcam).
Ubiquitination determination was carried out as the transfected cells were lysed using RIPA buffer containing 0.1% SDS to a final concentration of 0.2% SDS, and then subjected to identical operations as the immunoprecipitation detection. In addition, Western blot analysis was conducted using an anti-Ub antibody (dilution ratio of 1:1000, ab134953, Abcam).
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