Hrp anti mouse

For Western blot, cells were lysed with RIPA lysis buffer containing protease inhibitors (cOmplete; Roche) and phosphatase inhibitors (PhosSTOP; Roche). Lysates were incubated for 20 min on ice and spun down for 10 min at 14,000 revolutions per minute at 4°C. Protein concentrations were normalized using the DC protein assay (Bio-Rad). Samples were denatured by adding Laemmli sample buffer (Bio-Rad) with 5% β-mercaptoethanol (Sigma-Aldrich) and boiled at 95°C for 5 min before loading. Proteins were separated on a 12% mini-PROTEAN TGX (Bio-Rad) precast gel and transferred onto a polyvinylidene fluoride membrane using the iBlot2 system (Thermo Fisher Scientific). The primary antibodies were SPRED1 (#94063; Cell Signaling Technologies), p-ERK (#9101; Cell Signaling Technologies), ERK (#9102; Cell Signaling Technologies), and β-ACTIN (A2228; Sigma-Aldrich). Secondary antibodies were HRP anti-mouse and HRP anti-rabbit (Cell Signaling Technologies). Membranes were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Ras activity was measured by purifying GTP-bound Ras from cell lysates using Ras Activation Assay Biochem Kit (BK008; Cytoskeleton) and following the manufacturer’s instructions. Band intensity was quantified with ImageJ.