Rab25

Immunohistochemical studies were performed on paraffin-embedded tissue sections. Deparaffinization (immunohistochemistry protocol for paraffin-embedded tissue sections, Cell Signaling Technology, MA, USA) was initially carried out, followed by the blocking of nonspecific antigen binding sites by using a universal blocking buffer for one hour at room temperature. Tissue sections were incubated with primary antibodies Rabbit polyclonal RAB25 (Abcam) and Mouse monoclonal Snail (Cell Signaling Technology, MA, USA) for one hour at room temperature, followed by incubation with the respective HRP-tagged secondary antibody for one hour at room temperature. The sections were washed two times for five minutes each with PBST. Immunostaining was detected by using the diaminobenzidine (DAB substrate) method (Cell Signaling Technology, MA, USA) according to the manufacturer's protocol. The peroxidase activity was detected in a DAB working solution containing DAB chromogen concentrate and DAB diluent. The tissue sections were counterstained with Mayer's hematoxylin. The slides were washed with double-distilled water and PBS and mounted with coverslips by using Histomount (National Diagnostics, GA, USA) upon complete drying. The samples were assessed for positive and negative staining by imaging under bright-light microscopy.

“Cells were washed with PBS and incubated with RIPA (Cell Signaling Technology) lysis buffer on ice. The collected lysate was spun down, and a Bradford assay was used to determine the protein concentration in the samples. Around twenty micrograms of protein from each sample was run through a 12% polyacrylamide gel. Proteins were transferred to a nitrocellulose blotting membrane (Amersham Protan 0.2 μm, GE Healthcare Life Sciences) and were blocked for an hour at room temperature by using TBS (10 mM Tris-HCL, 150 mM NaCl, 0.1% Triton X-100, and pH 8.0) buffer containing 5% skim milk. The membranes were incubated with the following antibodies overnight on a shaker at 4°C. The membranes were then washed three times for five minutes each with TBST (TBS buffer with 0.05% Tween 20). The membranes were incubated with respective secondary antibodies for one hour at room temperature on a shaker.” Primary antibodies used were Mouse monoclonal β actin (Cell Signaling Technology, MA, USA), Rabbit polyclonal RAB25 (Abcam), Mouse monoclonal H-RAS (Thermo Fisher Scientific, MA, USA), Rabbit monoclonal E-cadherin (Cell Signaling Technology, MA, USA), Rabbit monoclonal claudin (Cell Signaling Technology, MA, USA), Goat polyclonal SNAIL (Santa Cruz Biotechnology, TX, USA), and Rabbit monoclonal IGF-1 Receptor (Cell Signaling Technology, MA, USA).