Protran

Cells were lysed on ice in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 1 mM NaF, 0.25% Na-deoxycholate, 1 mM Na₃VO₄, and 150 mM NaCl) containing a protease/phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA). Lysates were centrifuged, and the proteins in the supernatant were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted to nitrocellulose membranes (Protran; Schleicher & Schuell, Dassel, Germany). Membranes were incubated with antibodies specific for FAK (1:1000, Santa Cruz, Dallas, TX, USA), pY397-FAK (1:1000, Cell Signaling Technology, Danvers, MA, USA), FLAG (1:2000, Sigma), SHP1 (1:1000, BD Bioscience, NJ, USA), SHP2 (1:1000, BD Bioscience), PTPD1 (1:1000, Thermo Scientific), and PTP-PEST (1:1000, Cell Signaling Technology). The membranes were washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with secondary antibodies, and the results were visualized using an enhanced chemiluminescence system (Daeil Lab Inc., Seoul, Korea).

Cells were lysed and nuclear or whole cell extracts were prepared as described previously.^{19 (link)} Protein concentration in the supernatants was determined by the BCA method. The cellular extracts were electrophoretically separated using 7.5 or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Proteins were transferred to nitrocellulose membranes (Protran, Schleicher & Schuell, Keene, NH, USA) and HIF-1α protein was detected in nuclear extracts (30–40 μg) or in whole cell lysates (50–70 μg) using a monoclonal anti-HIF-1α antibody purchased from BD Biosciences Pharmingen (San Jose, CA, USA). Donkey anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a dilution of 1:2000. PLD1 and 2 proteins were detected in whole cell extracts (30–40 μg) using anti-PLD antibody.^{18 (link)} Goat anti-rabbit secondary antibody (KPL protein research products, Gaithersburg, MD, USA) was used at a dilution of 1:5000. Signals were visualized using the SuperSignal chemiluminescence substrate (Pierce, Rockford, IL, USA). Experiments were performed in duplicate and normalized with antibodies to topoisomerase II (Santa Cruz Biotechnology) for nuclear HIF-1α and to β-tubulin (Santa Cruz Biotechnology) for PLD and HIF-1α in whole cell lysate.