Ab37647

HEK293D cells were cultured and transfected using Lipofectamine 2000 (Thermo Fisher Scientific). The supernatant was collected and esRAGE in the supernatant was confirmed by ELISA (B-Bridge International K1009-1) and Western blot. The supernatant containing esRAGE was mixed in equal parts (100 μl) with recombinant HMGB1 (0.1 mg/ml, Sigma H4652) and incubated for 1.5 hours at 37 °C. Using Pierce Co-immunoprecipitation Kit (Thermo Fisher 26149), the esRAGE-HMGB1 complex is then pulled down with anti-RAGE (Abcam ab37647) antibody coupled resins.
The co-immunoprecipitated proteins were then separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-RAD). The membranes were blocked and then incubated either with anti-HMGB1 (Abnova H00003146-M08 or Abcam ab18256) or anti-RAGE (Abcam ab37647) antibodies overnight, washed and incubated with horseradish peroxidase-conjugated antibody. Bands were visualized by chemiDoc MP imaging system using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Immunofluorescence staining was performed as previously described (Wu et al., 2019b (link)). Briefly, tumor and animal tissues were fixed with paraformaldehyde, dehydrated, and cleared with a gradient of alcohol solutions and xylene, respectively. Consecutively, tissues were embedded in paraffin. Paraffin-embedded tissues were sliced continuously (3.5°C) using a HistoCore AUTOCUT system (Leica), mounted, dewaxed, and rehydrated. For antigen retrieval, sections (3–4 μm) were pretreated in a microwave oven with citric acid buffer (pH 6.0) for 12 min and then cooled to 25°C in deionized water. After washing with PBS for 5 min, sections were incubated with 5% normal goat and donkey serum at 37°C for 1 h at 25°C and then incubated with the primary antibody (1:800, ab37647, Abcam) at 4°C overnight. After washing at least thrice with PBS, sections were incubated with the following secondary antibodies: Alexa 633-conjugated donkey anti-rat antibody (1:500, Invitrogen), Alexa 596-conjugated goat anti-rabbit (1:500, Invitrogen), and Alexa 488-conjugated goat anti-mouse antibody (1:250, Invitrogen) for 1 h at 25°C. Afterward, cells were extensively washed with PBS, and their nuclei were labeled using 4′,6-diamidino-2-phenylindole (DAPI). Free-floating sections with positive immunofluorescence staining were captured and analyzed using a laser scanning confocal microscope (Zeiss).

Allicin (purity ≥ 98%) was purchased from Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, China). BSA (purity ≥ 98%) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fructose and nitro blue tetrazolium (NBT) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Aminoguanidine and Girard-T reagent were purchased from Aladdin Chemical Co. (Shanghai, China). An anti-RAGE antibody (ab37647) was purchased from Abcam (Shanghai, China).