Agarose beads

After transfection, cells were harvested and lysed with special lysis buffer (5 mM HEPES, pH 7.8, 100 μM sodium orthovanadate, and 0.1% Triton X-100). The lysates were sonicated and the presence of overexpressed protein was confirmed by performing SDS-PAGE and Western blot analysis. For immunoprecipitation, the cell lysates were treated with 1 μl monoclonal antibody for the respective protein and 10 μl agarose beads (Millipore, Billerica, MA) and incubated at 4°C for 4 h. After incubation, the immunoprecipitates were washed with LiCl wash buffer (2.1% LiCl, 1.6% Tris-HCl, pH 7.4) and NaCl wash buffer (0.6% NaCl, 0.16% Tris-HCl, 0.03% EDTA, pH 7.4), respectively, and sedimented at 12,000 × g for 1 min. The resulting pellets were then analyzed using SDS-PAGE and Western blot (WB) analyses.

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.

RAW264.7 cells were cultured in reduced bicarbonate DMEM plus 10% fetal bovine serum. The plasmids used for transfections were pcDNA3.1-mycPLD2-WT, pcDNA3.1-XGrb2 and pEGFP-C1-WASp. When cultured cells reached a confluence of 60%, they were transfected with the plasmid of interest. Transfections were performed using 5 µl Superfect (Qiagen, Valencia, CA, USA) in Opti-MEM media previously mixed in sterile glass test tubes. The DNA and Superfect were mixed in post-transfection media (without antibiotics), applied to cells, and then transfected for 36 h. After transfection, the cells were harvested and lysed with Special lysis buffer (5 mM HEPES, pH 7.8, 100 µM sodium orthovanadate, and 0.1% Triton X-100 and protease inhibitors (aprotinin 2 µg/ml and leupeptin 5 µg/ml). The lysates were sonicated and treated with 1 µl monoclonal antibody for the respective protein and 10 µl agarose beads (Millipore, Billerica, MA) and incubated at 4 °C overnight. After incubation, the immunoprecipitates were washed with LiCl wash buffer (2.1% LiCl, 1.6% Tris-HCl, pH 7.4) and NaCl wash buffer (0.6% NaCl, 0.16% Tris-HCl, 0.03% EDTA, pH 7.4), respectively, and sedimented at 12,000 × g for 1 min. The resulting pellets were then analyzed using SDS-PAGE and Western blotting (WB).