Anti per1

The TPBC and TNBC sections were hydrated and treated with 1.5% hydrogen peroxide for 1 h at 37°C, followed by treatment in 0.05 M citrate-buffered saline (pH 6.0) at 95°C for antigen retrieval. The TPBC and TNBC sections were then incubated in 5% goat serum (Gibco) for 1 h at 37°C prior to incubation with the primary antibodies (1:200 dilutions) anti-BMAL1, anti-PER1, anti-ASMT (Abcam), anti-CLOCK (Abcam, 1:1000), and anti-serotonin N-acetyltransferase N-terminal (Sigma-Aldrich, 1:200) for 1 h at 37°C and overnight at 4°C. Subsequently, the TPBC and TNBC sections were incubated with the biotinylated goat anti-rabbit IgG (Santa Cruze Biotechnology, 1:200) for 1 h at 37°C, followed by incubation with avidin-biotin peroxidase complex (1:200) and dehydration with ethanol. Data are presented as the percentages of positive areas of ASMT, NAT, CLOCK, BMAL1, and PER1 relative to the total area. Six uncontinuous fields were selected from one slide at a magnification of 200×.

Proteins were extracted following the procedure described previously [35 ]. The proteins were separated by SDS-PAGE on 8–15% polyacrylamide gels and subsequently electrically transferred to a PVDF membrane. After blocking with 5% (w/v) BSA in TBST at room temperature for 1 h, the membranes were then incubated with appropriate specific primary antibodies: anti-GPX1 (1:2000, Abcam, Cat#ab108427, Cambridge, MA, USA); anti-HA tag (1:1000, Cell Signaling, Cat# 3724, Danvers, MA, USA); anti-PER1 (1:200; Abcam, Cat# ab3443, Cambridge, MA, USA); anti-β-actin (1:1000, Bioworld, Cat# AP0060, MN, USA) at 4 °C overnight, followed by incubation with an HRP-conjugated secondary antibody (1:10000 Beyotime, Cat#A0208, Shanghai, China). Detection was performed using an enhanced chemiluminescence kit (Thermo Scientific, Hudson, NH, USA).

The amount of protein was determined using the BCA protein assay (Thermo Scientific, Rockford, IL, USA), and the same amounts of proteins were normalized for total protein. The protein samples were boiled in RIPA buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and protease inhibitor cocktail; Sigma-Aldrich, St Louis, MO, USA), separated by SDS–polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Non-specific binding was blocked with 5% skim milk in PBS-Tween20, and proteins were probed with anti-EAAC1 (Abcam, Cambridge, MA) at 1:1,000 dilution, anti-PER1 (Abcam) at 1:200, anti-PER2 (Abcam) at 1:1,000, anti-BMAL1 (Abcam) at 1:1,000, anti-CLOCK (Abcam) at 1:1,000 and anti-β-actin (Sigma-Aldrich) at 1:10,000 dilution. After a wash with PBS-Tween20, the horseradish peroxidase-labelled secondary antibodies were probed and detected with the ECL prime HRP detection kit (GE Healthcare, Piscataway, NJ, USA). We performed the quantification of the EAAC1 level using a serial dilution of ZT14 samples as the standard curve.