Pfkfb4

Interaction between endogenous SRC-3 and PFKFB4 was detected using the PLA technique^{34 (link)} using Duolink In Situ Red Starter Kit Mouse/Rabbit (Cat# DUO92101, Sigma) according to manufacturer’s instructions. Briefly, MDA-MB-231 cells were seeded in 35 mm glass bottom culture dish (Cat# P35G-0-14C, MatTek Corporation), and after reaching 80% confluency, cells were fixed followed by blocking for 1 hour using the Duolink Blocking Solution at 37°C. Cells were then incubated in presence of primary antibodies: SRC-3 (Rabbit Monoclonal, Cell Signaling) and PFKFB4 (Mouse Monoclonal, Origene), either alone or in combination. Following incubation, cells were washed and Duolink PLA PLUS and MINUS probes were added for 1 hour at 37°C. After washing off the unbound probes, cells were incubated first with the ligase enzyme followed by DNA polymerase enzyme to amplify the DNA circle. Finally cells were mounted using Duolink in Situ Mounting Media with DAPI, and analyzed by microscopy. Images were obtained using Zeiss Axio Observer A1 inverted microscope with N-Achroplan 100×/1.25 oil lens, Zeiss MRC5 camera, and AxioVision Rel.4.8 software.