Ultrasensitive s p kit

The NE‐PERTM Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) was used to isolate and collect cytosolic and nuclear fractions following the manufacturer's protocol. Cells were lysed in radioimmunoprecipitation assay (RIPA) lysate buffer, and cell lysates were incubated on ice for 30 minutes. Cell supernatants were collected, and protein concentrations were determined using bicinchoninic acid (BCA) protein quantitation (Beyotime). SDS‐PAGE electrophoresis was performed on proteins from cell lysate proteins and transferred to PVDF membranes. Membranes were incubated with primary antibody overnight at 4℃. The primary antibodies and secondary antibodies are shown in Table S2. Proteins in membranes were visualized using an enhanced chemiluminescence kit (BOSTER).
For IHC, mouse tumour sections were dewaxed and rehydrated. The antigen was retrieved under high pressure using citrate buffer (pH = 6.0). The Ultra‐sensitive S‐P kit (Maixin‐Bio) was used to block endogenous peroxidase activity and reduce non‐specific reactivity. Sections were then incubated with primary antibodies (shown in Table S2) at 4°C overnight. Mouse tumour sections were then incubated with secondary antibody and streptomycin avidin‐peroxidase using the Ultra‐sensitive S‐P kit, and the sections were visualized with DAB reagent (Maixin‐Bio).

Formalin-fixed paraffin-embedded tumor specimens were collected from the Department of Pathology at the First Hospital of China Medical University. Immunohistochemical staining was performed using the following antibodies: rabbit anti-RANK (sc-52951, 1:500, Santa Cruz Biotechnology, Santa Cruz, USA), anti-Cbl-b (sc-376409, 1:250, Santa Cruz Biotechnology), p-ERK (1:1000, Cell Signaling Technology, Danvers, MA, USA) and p-Akt (1:1000; Cell Signaling) using the biotin-streptavidin method (UltraSensitive S-P kit; Maixin-Bio, Shanghai, China) as described previously [36 (link)]. The evaluation of immunohistochemistry results was performed independently by two observers who had no prior knowledge of the clinical information or pathological parameters. The immunoreactivity was scored based on both intensity of staining (negative = 0, weak = 1, moderate = 2, strong = 3) and percentage of positive tumor cells (<10% = 0, 10%–50% = 1, >50% = 2) as reported previously. The final score was calculated by multiplying the single scores to obtain the intensity and percentage of positive cells (range from 0 to 6) [36 (link), 37 (link)]. The median expression score for both RANK and Cbl-b was 2, and this was used as a cut-off value, with a score of at least 2 considered positive.