Ab75766

The normal tissues (NT) from healthy individuals (n = 30) as well as peritumoral tissues (PT) and tumour tissues (TT) from HCC patients (n = 30) were fixed with 4% (w/v) paraformaldehyde for 15 min, washed five times with PBS, permeabilized with 0.1% (v/v) Triton X-100 (Sigma-Aldrich) for 5 min, embedded in paraffin, and cut into 5-μm sections. After deparaffinization, the histological sections were incubated at 100 °C for 15 min in sodium citrate buffer (10 mmol/L sodium citrate, 0.05% (v/v) Tween 20, pH 6.0) for antigen retrieval and treated with 3% (v/v) hydrogen peroxide (H₂O₂) at room temperature for 15 min to block endogenous peroxidase activity in the tissues. Next, the tissues were washed five times with PBS and blocked with 5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) and 1% (v/v) normal donkey serum (Sigma-Aldrich) for 1 h at room temperature. The tissues were incubated with a rabbit monoclonal anti-PHB1 primary antibody (1:300 dilution, ab75766; Abcam, MA, United States) for 16 h at 4 °C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1:1000 dilution; Santa Cruz, CA, United States) for 1 h. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and the tissues were mounted with neutral balsam mounting medium. The tissues were visualized using an inverted microscope (Nikon, Tokyo, Japan).

The interaction of PHB with AKT was assessed using a coimmunoprecipitation (co-IP) assay with mouse caudal epididymal sperm after incubated in BWW for 3 h in a 5% CO2 incubator at 37°C. Briefly, collected sperm were homogenized in 1 ml of cold lysis buffer (150 mmol l⁻¹ NaCl, 200 mmol l⁻¹ Tris-HCl, 1 mmol l⁻¹ ethylene diamine tetraacetie acid [EDTA] pH 8.0, 1% Triton X-100, 0.5% NP-40, 1 × proteinase inhibitor cocktail, 1 × phosphatase inhibitor cocktail) for 40 min on ice before centrifugation (14 000g, 15 min, 4°C). Total protein concentrations of the lysates were quantified using the Bradford method (Pierce Biotechnology, Rockford, IL, USA). Lysate samples with 1 mg of total protein were gently rotated at 4°C overnight while incubated with 5 μg of PHB antibody (ab75766, Abcam, Cambridge, MA, USA) or 5 μg rabbit IgG. The PHB- or IgG-targeted immune-complex was collected after incubation with 50 μl of protein A+G agarose beads (Santa Cruz Biotechnology) via gently shaking at 4°C for 4 h before centrifugation at 1500g for 2 min at 4°C. The precipitate was washed three times with ice-cold washing buffer, resuspended in 4× protein loading buffer, and boiled for 10 min to dissociate the immune-complex from the beads. The supernatant was collected by centrifugation and subjected to standard SDS-PAGE and immunoblot procedures as previously reported.5 (link)6 (link)