VALIDATION IMAGES
U-937 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20℃, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with phospho-NFKB p65 (Thr435) Antibody(bs-5661R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
Tissue/cell:Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum,C-0005) at 37°C for 20 min; Antibody incubation with (phospho-NFKB p65 (Thr435)) polyclonal Antibody, Unconjugated (bs-5661R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Tissue/cell:MCF7 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum,C-0005) at 37°C for 20 min; Antibody incubation with (phospho-NFKB p65 (Thr435)) polyclonal Antibody, Unconjugated (bs-5661R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
MCF-7 (H) cells were treated with or without hTNF-α (20 ng/ml) and Calyculin A (100 nM) for 15 min, 25 μg total protein per lane of cell lysates (see on figure) probed with phospho-NFKB p65 (Thr435) polyclonal antibody, unconjugated (bs-5661R) at 1:1000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.