Ras ep1125y

CRC cells (SW480 and SW620) were transfected with NNT-AS1 siRNAs or Control siRNA in 6-well plates. Seventy-two hours after transfection, the cells were washed twice with cold PBS, and 30 μL of RIPA (Solarbio, Shanghai, China) containing a protease inhibitor cocktail (Invitrogen, Carlsbad, CA, USA), a phosphatase inhibitor cocktail (Invitrogen, Carlsbad, CA, USA) and 2 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0 was added to each well. Following this, the cells were collected into a cold tube. Cell lysates were centrifuged at 12,000 g for 20 min at 4°C. Subsequently, the proteins in the lysates were separated on a 10% polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 10% non-fat milk for 4 h at room temperature and then incubated with primary antibodies for 1 h at 4°C. Antibodies against the following proteins were used: E-cadherin (24E10), vimentin (D21H3), p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E), GAPDH (D16H11) (from Cell Signaling Technology, Danvers, MA) and Ras [EP1125Y] (from Abcam, Cambridge, UK), followed by second antibody (zhongshanjinqiao, China). Protein bands were visualized using Super Enhanced Chemiluminescence Detection regents (Applygen Technologies, Beijing, China).

Following antibodies were used for western blotting: Actin (I-19), Ezrin (C-19), Radixin (C-15), Moesin (C-15), Merlin (B-12), MYPT1 (H-130), c-myc (9E10) from Santa Cruz Biotechnology; CPI-17 (E305) from Epitomics; Ezrin (3C12) from Neomarkers; Ezrin (3145), Radixin (C4G7), phospho-ERM (3149) from Cell Signaling Technology; Moesin (EPR2429(2)) from Abcam; phospho-NF2 S518 from US Biologicals; VSVg from Roche. For Ras, either the antibody from the Active Ras Pulldown and Detection Kit from Thermo Fisher Scientific was used, or Ras (EP1125Y) from Abcam (Figure 4C). For immunoblots of ERM (unless otherwise stated), a mixture of Ezrin (C-19), Radixin (C-15) and Moesin (C-15) antibody was used; for single Ezrin blots, the 3C12 antibody was used. Ezrin (3145), Radixin (C4G7) and Moesin (EPR2429(2)) were used for Figures 1D, 3A, 4A and Supplementary Figure S2. Staining of paraffin sections was performed using anti-CPI-17 from Millipore and HMB45 from DAKO. Ras antibody Y13-259 (Thermo Fisher Scientific) was used for the GEF assay.