Western blotting analysis using sirtuin 3 antibody (Cat#65604). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with sirtuin 3 antibody (Cat#65604, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using NaQ™ ECL Substrate Kit (Cat#716).
Western blotting analysis using sirtuin 3 antibody (Cat#65604). Sirtuin 3 expression in wild-type (WT) and sirtuin 3 (SIRT3) shRNA knockdown (KD) HeLa cells with 25 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with sirtuin 3 antibody (Cat#65604, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).
Immunohistochemistry was performed on paraffin-embedded human breast carcinoma using sirtuin 3 antibody (Cat#65604, 1:500). Antigen retrieval was done in sodium citrate buffer (pH 6.0). DAB was used for detection, with hematoxylin counterstaining. Images were acquired using a Nikon Ci-L Plus microscope (40× objective). Scale bar, 25 μm.
Immunocytochemical staining of HeLa cells using Sirtuin 3 antibody (Cat#65604, 1:1,000), Top panel: wild-type (WT); Bottom panal: Sirtuin 3 shRNA knockdown (KD). Nuclei were stained blue with DAPI; Sirtuin 3 was stained magenta with Alexa Fluor® 647. Scale bar, 20 μm.
Immunocytochemical staining of HAP-1 cells with Sirtuin 3 antibody(Cat#65604, 1:1,000) . Nuclei were stained blue with DAPI; Sirtuin 3 was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: Medium. Scale bar, 20 μm.
Validation of Sirtuin 3 knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HeLa cells were stained with Sirtuin 3 antibody (Cat#65604, 1:2,000) and analyzed using BD flow cytometer.
