Flow cytometric analysis of PFKFB3 expression in HT-1080 cells using PFKFB3 antibody (R021242, 1:2,000). Green, isotype control; red, PFKFB3.

Immunocytochemical staining of HT-1080 cells with PFKFB3 antibody (R021242, 1:1,000) . Nuclei were stained blue with DAPI; PFKFB3 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.

Western blotting analysis using PFKFB3 antibody (R021242). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with PFKFB3 antibody (R021242, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (1:20,000) respectively. Image was developed using ECL Substrate Kit.

Western blotting analysis using PFKFB3 antibody (R021242). PFKFB3 expression in wild type (WT) and PFKFB3 shRNA knockdown (KD) HT-1080 cells with 30 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with PFKFB3 antibody (R021242, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (1:20,000) respectively. Image was developed using ECL Substrate Kit.

Validation of PFKFB3 knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HT-1080 cells were stained with PFKFB3 antibody (R021242, 1:2,000) and analyzed using BD flow cytometer.