Anti spry4

Proteins were isolated after transfection with siRNAs using RIPA buffer (SIGMA-ALDRICH) containing 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, phosphatase inhibitors and protease inhibitors. The protein concentration was measured using protein assay dye reagent concentrate (BioRad), and 30 µg protein was loaded onto 10% Mini-PROTEAN® TGX™ Precast Gels (Bio-rad), unless otherwise specified (Supplementary Fig. S5). After SDS-PAGE, the proteins were blotted onto a PVDF membrane, and the membrane was blocked in TBST with 5% skim milk before incubating with primary antibody overnight at 4 °C. An HRP conjugated secondary antibody was used, and the proteins were detected using the ImageLab machine (BioRad). Optical density (OD) of the protein bands was determined by using Image Studio Lite. Primary antibodies used in this study were: Anti-β-actin (Abcam, 1:1000), Anti-phospho-ERK1/2 (Cell Signaling, 1:2000), Anti-ERK1/2 (Cell Signaling, 1:1000), Anti-phospho-Akt (ser473) (Cell Signaling, 1:2000), Anti-Akt (Cell Signaling, 1:1000), Anti-SPRY4 (Abcam, 1:1000). HRP-conjugated anti-rabbit (Cell Signaling, 1:2500) was used as secondary antibody.