Anti c met

Cellular proteins were extracted and separated in SDS-PAGE gels, and Western blot analyses were performed according to standard procedures. The membranes were immunoblotted with the following antibodies: anti-c-Met (1:500, Abcam), anti-Mecp2 (1:500, Abcam), and anti-Tublin (1:1000, Abcam). As a loading control, GAPDH detection was performed with primary antibody (1:5000, Abcam).

WB was performed using conventional WB procedures. Primary antibodies were purchased from CST (Cell Signaling Technology, Danvers, MA, USA), including anti-HGF anti-phospho Met (#3133), anti-cMet (#8198), anti-Akt (#2920), anti-phospho-Akt (ser473, #4060), anti-pPDGFR (Tyr1009, #3124), and anti-phospho-Erk1/2 (Thr202/Tyr204, #4370).
Human cytokine antibody array (Abcam, Cambridge, USA, ab133998) was performed according to the manufacturer's instruction manual.
For MET activation and neutralization experiments, RKO cells were seeded in 24-well plates at a density of 40,000 cell per well in culture medium containing 10% FBS. One day before treatment, the wells were refreshed with medium containing 2% FBS. CAF CM diluted with culture medium (2% FBS) at 1:2 was added to the cells for 20 min at 37°C. Alternatively, the HGF neutralizing antibody was added to the diluted CAF CM and incubated for 1 h before treatment. The cells were lysed with a RIPA-containing phosphatase inhibitor cocktail for preparation of the WB samples.

The HGF, cMET and EGFR expression was assessed by western blotting analysis and samples were normalized to GAPDH. Protein extraction was blocked with PBS-5% fat-free dried milk at RT for 1 h and incubated at 4 °C overnight with anti-HGF (1:1,000, Abcam), anti-EGFR (1:5,000, Abcam), anti-cMET (1:1,000, Abcam), anti-p-cMET (1:1,000, Abcam), anti-CD63 (1:2,000, Abcam), anti-TSG101 (1:1,000, Santa Cruz), anti-Alix (1: 1,000, Santa Cruz), anti-F4/80 (1:1,000, Abcam), anti-desmin (1:1,000, Immunoway), anti-α-SMA (1:1,000, Santa Cruz) and anti-GAPDH (1:3,000, Santa Cruz) antibodies, respectively.

Western blot analysis was performed as described previously [16 (link)]. The primary antibodies were the following: anti-E-cadherin (1:1000, Cell Signaling Technology, USA), anti-N-cadherin (1:1000, Cell Signaling Technology), anti-Vimentin (1:1000, Abcam, USA), anti-activated caspase-3 (1:1000, Cell Signaling Technology), anti-total caspase-3 (1:1000, Cell Signaling Technology), anti-c-Met (1:1000, Abcam), and anti-GAPDH (1:1000, Santa Cruz Biotechnology, USA).

Following antibodies were used for IB analysis. Anti-USP21 (catalog MA5-34953), anti-FOXD1 (catalog PA5-27142) were purchased from Invitrogen. Anti-TAZ (catalog ab84927), anti-C/EBPβ (catalog ab32358), anti-c-MET (catalog ab74217), anti-ALDH1A3 (catalog ab129815), anti-OLIG2 (catalog ab109186), anti-Flag (catalog ab205606), anti-Myc (catalog ab32), anti-HA (catalog 236632), and GAPDH (catalog ab8245) were purchased from Abcam. Anti-CD44 (catalog 3570), anti-p-STAT3 (catalog 9145), anti-STAT3 (catalog 9139), anti-mouse IgG (catalog 5415), anti-rabbit IgG (catalog 3900), and anti-GST (catalog 2624) were purchased from Cell Signaling Technology.
Samples or cells with indicated treatments were lysed with cell lysis buffer (Beyotime, catalog P0013) with protease Cocktail Inhibitor (MCE, catalog HY-K0010) at 4 °C for 30 minutes. The lysates were centrifugated at 12,000 rpm for 20 min. Cell lysates were subjected to SDS-PAGE, transferred onto a polyvinylidenedifluoride membrane (Roche, catalog 03010040001), followed by incubation overnight at 4 °C with primary antibodies. The membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Proteins were then detected with the enhanced chemiluminescence methods.