Fluorophore conjugated secondary antibody

Ascorbic acid was purchased from Sigma (St Louis, MO, USA); chemically defined lipid concentrate from Invitrogen (Carlsbad, CA, USA); endothelial growth basal medium-2 (EBM-2) from Lonza (Walkersville, MD, USA); human basic fibroblast growth factor (bFGF) from Cell Signaling Technology (Danvers, MA, USA); hydrocortisone from Fisher Scientific (Pittsburg, PA, USA); fetal bovine serum (FBS) from Hyclone (Logan, UT, USA); penicillin/streptomycin from Cellgro Mediatech, Inc. (Manassas, VA, USA); type I collagen from R&D System (Minneapolis, MN, USA); TritonX-100 and bovine serum albumin (BSA) both from Sigma-Aldrich (St Louis, MO, USA); primary antibodies for occludin and ZO-1 (both diluted 1:100; Abcam Cambridge, MA, USA); fluorophore-conjugated secondary antibody from Proteintech Group (diluted 1:200; Chicago, IL, USA); and 4′,6-diamidino-2-phenylindole from Sigma-Aldrich.

NPSCs which were seeded on glass coverslips were washed with PBS, and fixed by 4% paraformaldehyde for 15 min. Cells were then permeabilized with 0⋅5% Triton X-100 (Beyotime) for 15 min at room temperature (for staining of Tie2, the permeabilization was not performed), and blocked with goat serum albumin for 1 h. Next, samples were washed with PBS and incubated with the mixture of mouse anti-HSP90 antibody (1:200, Santa Cruz Biotechnology) and rabbit anti-phosphorylated MLKL (P-MLKL) antibody (Ser358, 1:200, Affinity Biosciences, OH, United States), the rabbit anti-HSP70 antibody (1:500, ABclonal, Wuhan, China), or rabbit anti-Tie2 antibody (1:100, Proteintech Group, Wuhan, China) at 4°C overnight, followed by incubation with fluorophore-conjugated secondary antibody (1:200, Proteintech Group). Finally, the nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI). Fluorescence images were observed using fluorescence microscope (Olympus IX71).

Immunofluorescent staining assays were performed to assess nuclear export of AR, sub-cellular localization of PIASs, SUMO1/3, MDM2 and molecular co-localizations. After transfection, cells were fixed with 3% formaldehyde for 30 min and then permeabilized with 0.1% Triton-100/PBS for 10 min. After being pre-blocked with 1% BSA/PBS, samples were incubated with indicated primary antibodies for 2 h, followed by incubation with fluorophore-conjugated secondary antibodies (Proteintech Group). Nuclei were counter-stained with 4′,6- diamidino-2-phenylindole (DAPI) after secondary antibody incubation. Cells were examined by fluorescence microscopy (Olympus).