Revert 700 total protein stain solution

Whole-cell lysates and immunoblotting were performed as described elsewhere (19 (link), 20 (link)). Blots were probed with the following antibodies obtained from Santa Cruz or CST (Danvers, MA, USA): phospho-ERK: Cat# sc-7383, RRID AB_627545, 1:1000; ERK: Cat# sc-514302, RRID : AB_2571739, 1:1000; SHP2: Cat# 3397, RRID: AB_2174959, 1:1000; PDGFRβ: Cat# sc-374573, RRID: AB_10990921, 1:100; pan-RAS: Cat# sc-166691, RRID: AB_2154229, 1: 200; GAPDH: Cat# sc-47724, RRID: AB_627678, 1:2000; anti-rabbit: Cat# 926-32211, RRID: AB_621843; Cat# 926-926-68071, RRID: AB_10956166; anti-mouse: Cat# 926-32210, RRID: AB_621842, Cat# 926-680707, RRID: AB_10956588; all 1:10,000, LiCOR (Bad Homburg, Germany). Primary antibodies were diluted in Intercept/TBS blocking solution (LiCOR) supplemented with 0.2% Tween-20, secondary antibodies were diluted in TBS supplemented with 0.1% Tween-20. Total protein staining was performed using Revert 700 Total Protein Stain Solution according to the manufacturer’s protocol (LiCOR). Densitometry was performed using Empiria Studio 1.2 (LiCOR).

Protein samples (5 μg each) were loaded on 4–20% gradient gel (Genscript, NJ, USA). After the electrophoresis run, the gel transferred to PVDF membrane using wet methods. The membrane was hydrated using 100% methanol for 30 s and washed with tris-buffered saline, pH7.4 (TBS) for 5 min at room temperature with gentle shaking. The membrane was stained with Revert 700 total protein stain solution (LI-COR, Inc., Lincoln, NE, USA) and incubated for 5 min at room temperature with washing solution (LI-COR, Inc., Lincoln, NE, USA). The membrane was then washed with Revert 700 Wash Solution n (LI-COR, Inc., Lincoln, NE, USA), rinsed two times for 30 s at room temperature with gentle shaking. The membrane was then briefly rinse with ultrapure water. For visualizing the membrane, the membrane was imaged in the 700 nm channel using Image Studio Lite Ver. 5.2 (LI-COR, Inc., Lincoln, NE, USA).