Anti hmgb2

Cells and exosomes were lysed and collected in a protein lysate, and the concentration was measured using a BCA kit (Thermo Fisher Scientific, 23327). Protein samples (30 μg) were electrophoresed, transferred to a nylon membrane and blocked with a blocking buffer. Finally, the cells were incubated with the primary antibody at 4 °C overnight. The antibodies used were anti-HMGB3 (cat:D160490, Sangon Biotech, China), anti-flotillin-1 (cat:ab41927, Abcam, USA), anti-actinin-4 (cat:ab108198, Abcam, USA), anti-Alix (cat:ab186429, Abcam, USA), anti-CD9 (cat:ab92726, Abcam, USA), anti-GAPDH (cat:10494-1-AP, Proteintech, China), anti-E-cadherin (cat:A3044, ABclonal, China), anti-Vimentin (cat:10366-1-AP, Proteintech, China), anti-N-cadherin (cat:A3045, ABclonal, China), anti-HMGB1 (cat:10829-1-AP, Proteintech, China), anti-HMGB2 (cat:14597-1-AP, Proteintech, China) and anti-HMGB4(cat:12787-1-AP, Proteintech, China). Following incubation with a goat anti-rabbit secondary antibody, the immunoreactive proteins were detected with ECL western blotting detection reagents (Millipore, WBKLS0500).

Paraffin sections, providing a clear and comprehensive view of the urethra, hindgut, URS, and rectourethral fistulas, were chosen for staining. Subsequently, the sections underwent deparaffinization and antigen retrieval. Blocking was carried out with serum (ZSGB-BIO, Beijing, China) at 25 °C for 1 hour, followed by overnight incubation at 4 °C with primary antibodies. The primary antibodies used were as follows: Anti-Pcsk9 (1:100), anti-Hmgb2 (1:100), and anti-Sod1 (1:100), all sourced from Proteintech (Wuhan, China). After PBS washes, the sections were incubated at 25 °C in the dark for 1 hour with fluorescent secondary antibodies: goat anti-rabbit 488 (1:100; Proteintech). Following PBS washes, the sections were stained with DAPI solution (Servicebio Technology, Wuhan, China) for 10 minutes. Finally, the sections were mounted using an anti-fluorescence fading medium (Solarbio, Beijing, China), and images were captured using a laser-scanning confocal microscope (LSM 880, Zeiss, Oberkochen, Germany).