Src 3

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.

The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG. ChIP assays were performed according to an EZ ChIP kit (Millipore) with some modification^{35 (link)}. Briefly, MDA-MB-231 cells were grown in 15 cm dishes until 80% confluent. For glucose stimulation, cells were glucose-deprived for 3 hours by incubating in glucose-free DMEM supplemented with 10% FBS, followed by 4 hours stimulation with 5mM or 25mM glucose. Cells were crosslinked in 1% formaldehyde and quenched with 125mM glycine. Chromatin was sheared by sonication using a Branson Sonifier, precleared with control IgG antibodies and agarose beads (Millipore), and then immunoprecipitated with IgG (control), SRC-3, pSRC-3-S857 and ATF4 antibodies. DNA fragments were eluted from beads followed by reverse-crosslinking and purified DNA was used in qPCR reactions using SYBR green (Applied Biosytems) to determine the promoter occupancy. Melt curve analysis was performed to verify all SYBR green reactions produced a single PCR product.