Odyssey scanner and software

For biotinylation assays, cells were serum‐starved and pretreated with low levels of 10 nM lactacystin and 100 nM concanamycin inhibitors for 1 hour before chilling on ice and biotinylation for recycling assays, as described previously.8, 61 After biotinylation, cells were stimulated with 0.5 nM HGF at 37°C in the presence of inhibitors for 7 minutes to allow internalization. Cells were placed on ice, stripped with reducing reagent (100 mM sodium 2‐mercaptoethanesulfonic acid [MesNa] in 50 mM Tris‐HCl [pH 8.6], 100 mM NaCl, 1 mM EDTA and 0.2% BSA) to remove noninternalized biotinylated proteins, and then returned to 37°C. To determine percentage of internalized proteins that recycled, cells were returned to ice, subjected to a second reduction with MesNa prior to lysis, recovered with NeutrAvidin‐agarose beads, and immunoblotted for detection of Met levels. Percent recycled Met was determined by quantifying immunoblots (n = 3) using the LI‐COR Odyssey scanner and software (LI‐COR Biosciences). The cycloheximide, lactacystin, sodium 2‐mercaptoethanesulfonic acid and iodoacetamide were purchased from Sigma. EZ‐Link Sulfo‐NHS‐SS‐Biotin and NeutrAvidin were obtained from Pierce Chemicals. Lactacystin and concanamycin were from Calbiochem.

The cells were collected and lysed in lysis buffer on ice, and the proteins were quantified using a Pierce BCA Protein Assay kit (Thermos Fisher Scientific, Inc., Waltham, MA, USA). Cell lysates were separated by 8-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene difluoride membranes. The membranes were subsequently incubated with the following primary antibodies overnight at 4 °C: E-cadherin (1:1000; cat. no. 3195; Cell Signaling Technology, Inc.), α-SMA (1:1000; cat. no. 4691; Cell Signaling Technology, Inc.), and vimentin (1:1000; cat. no. 5741; Cell Signaling Technology, Inc.). The membranes were washed three times with TBS/T and then incubated for 1 h in IRDye®680RD goat anti-rabbit immunoglobulin G (H + L) diluted at 1:5000 in TBST. Protein levels were visualized and quantified using the LI-COR Odyssey scanner and software (LI-COR Biosciences).

Total protein fraction was extracted by dissolving the cell pellet in 2 × Laemmli buffer (0.125 M Tris–HCl (pH 6.8), 20% glycerol, 0.2% 2-mercaptoethanol, 0.004% bromphenol blue, 4% SDS) and incubated at 95 °C for 20 min. Five µl of the total protein fraction (corresponding to 25–35 µg of proteins) was loaded per well in a 10% SDS-PAGE gel. Gels were run at 160 V for 75 min, and the proteins were transferred to nitrocellulose membranes using the Trans-Blot Turbo™ Transfer system (Bio-Rad, Hercules, CA, USA). The membranes were incubated in blocking buffer (3% Fish Gelatin in PBS-Tween 0.1% (PBS-T) at RT for 1 h prior to O.N incubation with primary antibodies in the blocking solution (see Table S1 for antibody details). Membranes were then washed 3 times with PBS-T (10 min), incubated with the appropriate secondary antibodies, either 680RD-conjugated or 800 W-conjugated (LI-COR Lincoln, NE, USA) (see Table S1) and finally washed 3 times (10 min) with PBS-T. Visualization and quantification were carried out with the LI-COR Odyssey scanner and software (LI-COR Lincoln, NE, USA). At least three independent experiments were analyzed for SDS-PAGE.

Cell extracts were prepared by lysing cells in 50 mM Tris (pH 7.6), 150 mM NaCI, 1% Triton X-100 and protease inhibitors for 30 min at 4 °C followed by centrifugation at 14,000 rpm for 15 min. The protein concentration of lysates was determined with the Bradford solution (Bio-Rad Laboratories, CA, USA) using bovine serum albumin as a standard. For western blotting, cell extracts were resolved on 10% acrylamide sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using the Mini-Protean system (Bio-Rad Laboratories, CA, USA) followed by transfer onto nitrocellulose membranes using the Mini Transblot wet transfer system (Bio-Rad Laboratories, CA, USA). Membranes were incubated in blocking solution (PBS+5% milk, 0.1%Tween-20) for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C and washed three times with PBS+0.1%Tween-20 (PBST). After secondary antibody incubation for 1 h at room temperature and washing three times with PBST, the blots were scanned using the LI-COR Odyssey® scanner and software (LI-COR Biosciences) at 169 µm.

Cells were lysed with RIPA buffer (Thermo Scientific) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentration was determined using the BSA Protein Assay (Thermo Fisher Scientific). Western blots were performed using NuPAGE 4–12% Bis-Tris Protein Gels (Invitrogen) as described before [26 (link)]. Briefly, gels were run in MES buffer (Invitrogen) and wet transferred onto the nitrocellulose transfer membrane. The following primary antibodies were used: PDCD4 (Cell Signaling, 9535), and ß-actin (Cell Signaling, 3700). Goat anti-rabbit IgG (H + L) 800 CW or goat anti-mouse (H + L) 680RD was applied for 45 min at room temperature (1: 25000, LI-COR) before washing with PBS containing Tween 20. Blots were imaged using an Odyssey Infrared Imaging System Scan and quantification was carried out with the LI-COR Odyssey® scanner and software (LI-COR Biosciences).