Ab237715

NPC cells were lysed in RIPA lysis buffer (Meilun Biotechnology Co., Ltd., Dalian, China) containing protease inhibitor cocktails (Fudebio, Hangzhou, China). Then, total protein from different samples (30 μg/per lane) was separated by SDS-PAGE and transferred onto 0.22 μM PVDF membranes (Amersham Bioscience, Piscataway, NJ, United States). After that, the membranes were blocked with 5% skimmed milk in TBST for 1 h and incubated with specific primary antibody overnight at 4°C. After being washed with TBST, the membranes were incubated with the secondary antibody for 1 h at room temperature. Next, the protein bands were developed using an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). Antibodies were listed: anti-E-cadherin (Abcam, ab76319, 1:2,000), anti-Vimentin (Abcam, ab92547, 1:2,000), anti-PIK3R1 (Abcam, ab191606, 1:1,000), anti-ERBB2 (Abcam, ab237715, 1:1,000), anti-GAPDH (Abcam, ab8245, 1:3,000), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719, 1:5000), and goat anti-rabbit IgG H&L (HRP) (Abcam, ab205718, 1:5,000).

We used tissue microarrays (F048Br01a, Bioaitech, China) comprising 24 BC tissues of varying stages and grades, along with their corresponding adjacent tumor tissues. IHC staining and scoring were conducted following a previously described protocol [20 (link)]. In summary, following the removal of paraffin, rehydration, and microwave antigen retrieval, the slides were left to incubate with antibodies overnight at a temperature of 4 degrees Celsius. Anti-ERBB2 (ab237715, Abcam), anti-SDC1 (ab130405, Abcam), and anti-MMP14 (ab51074, Abcam) were each diluted to concentrations of 1:2000, 1:1000, and 1:800, respectively. Afterward, the slides were subjected to secondary antibodies at ambient temperature for a duration of 30 minutes. Then, they were stained using DAB substrate and subsequently counterstained with hematoxylin.

Tissues and cells were lysed using RIPA lysis buffer (Sigma Aldrich) and the total proteins were quantified using PierceTM BCA protein assay kit (Thermo Fisher Scientific). Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene difluoride membranes. The membranes were blocked using 5% bovine serum albumin (BSA) for 1 h, and incubated overnight at 4 °C with the primary antibodies, including anti-Hsp90 (Abcam ab13492, 1:500), anti-PD-L1 (Abcam ab205921, 1:500), anti-HER2 (Abcam ab237715, 1:500), anti-p-HER2 (Abcam ab108371, 1:500), anti-AKT (Abcam ab131168, 1:1000), anti-p-AKT (Abcam ab38449 1:500), and tubulin as internal control (Abcam ab6160, 1:1000). Next, the membranes were washed using TBST for five times and then incubated at room temperature with HRP-conjugated secondary antibodies for 1 h. Finally, the membranes were visualized and the protein intensities were detected using ECL chemiluminescence reagents (Pierce).