Abs152589

The immunohistochemistry (IHC) assay followed a previously published protocol (Jin et al., 2018 (link)). Briefly, paraffin-embedded breast tissue slides with a thickness of 5 µm were incubated overnight at 4 °C with anti-PIMREG (1:50; abs152589; absin, Shanghai, China). Subsequently, the slides were incubated with the secondary antibody at room temperature for 1 h. Then, the slides were incubated in diaminobenzidine (DAB) and counterstained with hematoxylin. Images of the slides were captured under a microscope.

To examine the protein expression of PIMREG in cells and BC tissues, a western blot assay was performed based on a previously published protocol (Xie et al., 2022 (link)). Briefly, the cells or BC tissues were lysed using RIPA reagent (Beyotime, Shanghai, China) and boiled for 10 min. Subsequently, the total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% nonfat milk diluted with Tris-buffered saline-Tween (TBST) at room temperature for 1 h and incubated overnight at 4 °C with the primary antibody: anti-PIMREG (1:500; abs152589; Absin Bioscience, Inc., Shanghai, China). The next day, the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature for 1 h. The Western blots were visualized using an ECL detection kit (Proteintech Group, Inc., Rosemont, IL, USA) for chemiluminescence. The density of the protein bands was quantified using Image J software (Pierce, Rockford, IL, USA), and the values was normalized to β-Actin.