Ab201212

Total proteins were obtained from HCC cells using RIPA buffer (Beyotime, Shanghai, China) and quantified by Enhanced BCA Protein Assay Kit (Beyotime). Then, 20 μg proteins were undergoing SDS-PAGE and transferred to the PVDF membrane (Millipore, Billerica, MA, United States). After blocking, the membrane was incubated overnight with primary antibodies at 4°C. The next day, secondary antibody incubation was performed at room temperature for 1–2 h. Enhanced chemiluminescence (ECL) reagent (Millipore) was used for luminous reaction and western blot was photographed by Amersham Imager 680 (GE Healthcare Life Sciences, Pittsburgh, PA, United States). The used primary antibodies: HIF-1α (ab1, Abcam, Cambridge, MA, United States), HIF-2α (ab199, Abcam), RNF146 (ab201212, Abcam), β-tubulin (10094-1-AP, Proteintech, Wuhan, China), PTEN (ab267787, Abcam). p-AKT (Ser473, 66444-1-Ig, Proteintech), AKT (60203-2-Ig, Proteintech), p-mTOR (Ser2448, 67778-1-Ig, Proteintech), mTOR (66888-1-Ig, Proteintech), Ubiquitin (ab7254, Abcam).

The embedded-paraffin tissues were sectioned with a thickness of approximately 4–5 μm. Next, paraffin sections were deparaffinized in xylene and rehydrated with ethanol, and used 0.3% hydrogen peroxide to block endogenous peroxidase activity. Finally, the sections were incubated with RNF146 antibody (ab201212, Abcam) and a specific secondary antibody. The intensity score and positive rate score were previously described (Shen et al., 2018 (link)). The IHC score (intensity score × positive rate score) ≥4 was defined as high expression.

The animal studies were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University. Male BALB/c nude mice (4 weeks old) were obtained from the Vital River Laboratory Animal Technology Co. (Beijing, China). 5 × 10⁶ HCCLM3 cells stably transfected with RNF146 shRNA or the negative control were respectively injected into the right armpits of five mice, and subcutaneous tumor size was assessed every week. The mice were sacrificed 4 weeks after injection and the xenograft tumors were removed for IHC with Ki-67 antibody (27309-1-AP, Proteintech) or WB with RNF146 antibody (ab201212, Abcam).