Ab195289

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).

The myocardial tissue sections were baked at 60°C for 1 h and dewaxed in xylene for 30 min. Subsequently, the sections were dehydrated with gradient alcohol of 95, 80, and 75% for 1 min/each, followed by incubation in 3% H₂O₂ (84885, Sigma-Aldrich, San Francisco, CA, United States) at 37°C for 30 min. Thereafter, the myocardial tissue sections were placed in 0.01 M citrate buffer, boiled at 95°C for 20 min, and then allowed to cool down to room temperature. After being sealed in normal goat serum at 37°C for 10 min, the sections were probed with rabbit anti-mouse monoclonal antibody USP22 (ab195289, dilution ratio of 1:100, Abcam, Cambridge, United Kingdom) overnight at 4°C. The sections were then reprobed with rabbit secondary antibody (ab6721, dilution ratio of 1:2,000, Abcam) for 30 min at room temperature. Next, the sections were treated with streptavidin–biotin–peroxidase complex for 30 min and stained with 3 mL of diaminobenzidine (DAB; DA1010; Beijing Solar Science and Technology Co., Ltd.) for 5–10 min. The sections were subsequently counterstained with hematoxylin, sealed, and fixed with neutral resin. Positive cells were represented as brown areas. Three areas of each sample in the experiment were randomly captured using a camera (Leica, Wetzlar, Germany) and analyzed using the ImageJ software.

The collected cells were lysed using a solution containing RIPA lysis buffer, phosphatase inhibitors, and protease inhibitors (Beyotime, China). The BCA reagent (Beyotime, China) was used to measure protein concentrations. Commensurable amounts of protein were separated using SDS-PAGE, transferred to a membrane, and incubated with various antibodies. Finally, the data were acquired using image Lab 5.2.1. Antibodies used were: BLIMP1 (ab243146, Abcam, 1:1000), USP22 (ab195289, Abcam, 1:1000), USP33 (ab237510, Abcam, 1:1000), SPI1 (ab227835, Abcam, 1:1000), PD-L1 (ab205921, Abcam, 1:1000), GAPDH (#5174, Cell Signaling Technology, 1:1000), HRP-linked anti-rabbit IgG (#7074, Cell Signaling Technology, 1:3000), and HRP-linked anti-mouse IgG (#7076, Cell Signaling Technology, 1:3000). Uncropped and unprocessed scans of blots are included in a Source Data file.