Ab137429

Cells were lysed in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Abcam, Cambridge, UK). Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Anti-NSD1 (sc-130470; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NSD2 (ab137429; Abcam), anti-NSD3 (ab137430; Abcam), anti-H3K36me2 (ab176921, Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab181602; Abcam) antibodies were used as the primary antibodies. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h and visualized by enhanced chemiluminescence (ECL) reagent (Millipore, Billerica, MA, USA).

Cervical tissue TMAs were prepared as described previously. Two-millimeter core TMAs were constructed from each of the collected cervical tissues that were marked by pathologists in Chongqing Medical University. Immumohistochemistry analyses were performed using anti-WHSC1 (ab137429; Abcam) antibodies. Immunohistochemical staining was performed on TMA sections according to the manufacturer’s protocol. We used a metastatic carcinoma with high expression of WHSC1 as a positive control in immunohistochemistry. Protein expression was scored and quantified by two independent pathologists blinded to the outcome of the cases.