Ab183506

We collected a total of 182 progesterone receptor positive human endometrial tissue samples, 107 stage III-IV cancer tissue of which had accompanying follow-up information, and 75 cancer-adjacent endometrial tissue samples from archives of paraffin-embedded tissues between May, 2011 and May, 2014 at the Department of Pathology of Peking Union Medical College Hospital. The follow-up was performed until May 30, 2019. The pathological diagnoses were reconfirmed by a pathologist. The project was approved by the Ethical Committee (Peking Union Medical College Hospital), and informed consent was acquired from patients or family members. IHC was performed as previously described (22 (link)). Anti-antibody (AKT1 1:250, Abcam, ab235958; PR 1:100, Abcam, ab32085; PBK 1:100, Abcam, ab75987; BIRC5 1:800, Abcam, ab469; AURKA 1:100, Abcam, ab52973; GTSE1 1:50, Abcam, ab103232; KNSTRN 1:50, Abcam, ab122769; PSMB10 1:100, Abcam, ab183506) was used for IHC. The scoring details have been described previously (23 (link)).

Protein samples were extracted using radioimmunoprecipitation assay (RIPA). The protein concentration of each extract was determined using a bicinchoninic acid protein assay kit (Pierce Chemical). Western blot analyses were performed using anti-LMP10 antibody (ab183506, Abcam), anti-phospho–IκBα (Ser32/36) (5A5, #9246, CST) and anti-IκBα (#9242, CST) antibodies, anti-phospho–NF-κB p65 (Ser536) (93H1, #3033, CST) and anti-NF-κB p65 (C22B4, #4764, CST) antibodies, with anti-GAPDH antibody (60004-1-lg, Proteintech) as control. Quantifications were performed with Image J software.

PBMCs (2 × 10⁷ cells/sample) were lysed in radio-immunoprecipitation assay (RIPA) buffer (Bestbio, Shanghai, China). Immunoblots were performed using polyclonal rabbit anti-human/mouse LMP2 (ab242061, Abcam), LMP7 (ab180606, Abcam), LMP10 (ab183506, Abcam), β-actin (ab179467, Abcam), GAPDH (ab181602, Abcam), p-STAT1 (Cell Signaling), STAT1 (Cell Signaling), p-STAT3 (Cell Signaling), and STAT3 (Cell Signaling) antibodies. Semi-quantitative evaluation of protein levels was performed by comparing band intensity between the protein of interest and the housekeeping controls, GAPDH or β-actin. The level of phosphorylated proteins was measured based on the ratio of phosphorylated protein to total protein.