Anti phospho met tyr1234 1235

Ears were collected, fixed in 4% PFA at 4°C overnight and washed. Then, they were cryoprotected in 30% and embedded in Tissue-Tek OCT (Sakura). 20- or 8-μm thick OCT sections were thawed for 30 minutes at room temperature, fixed in 4% PFA for 10 minutes and permeabilized with 0.3% Triton X-100 in PBS. After 30 minutes in blocking buffer (0.5% BSA, 5% donkey serum, 0.3% Triton X-100, 0,1% NaN3), slides were incubated with primary antibodies overnight at 4°C. The next day slides were washed with 0.3% Triton X-100 in PBS for 30 minutes and appropriate alexa secondary antibodies 488/555 were added 1:500 in blocking buffer for 1 hour at RT. After that, slides were washed with 0.3% Triton X-100 in PBS for 30 minutes and mounted.
The following antibodies were used: either unlabelled or biotin-labelled Ly6G (Biolegend), anti-F4/80-APC (Invitrogen), anti-MET (Abcam), anti-Phospho-MET (Tyr1234/1235) (Cell Signaling Technology), anti-CD206 (Biolegend). Images were acquired on a Zeiss LSM880 and analysed using Imaris. Quantification of Ly6G⁺ and F4/80⁺ cells was performed using ImageJ software. 3D rendering and visualisation were also generated using the Imaris cell imaging software.

Western blot analysis was performed using an extraction buffer as described [44 (link)]. MKN-45, MKN-28 and U87 cells were cultured in serum-free medium overnight, then treated with the indicated dose of peptide CM 7 for 24 h at 37 °C after stimulated without or with HGF for 15 min. Next, total cell lysates were separated by 12% SDS-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were probed with following primary antibodies: anti-phospho-Met (Tyr1234/1235, (Cell Signaling Technology, Beverly, MA, USA), anti-Met (Affinity, USA), anti-phospho-AKT (Ser473, (Affinity, USA)), anti-AKT (Affinity, USA), anti-phospho-Erk1/2 (Thr202/Tyr204, (Affinity, USA)), anti-Erk1/2 (Affinity, USA), anti-E-cadherin (WanleiBio, Shengyang, China), and anti-GAPDH (Multi Sciences, Hangzhou, China). Then, horseradish-peroxidase-conjugated anti-rabbit IgG was used as a second antibody. The immunoreactive proteins were exposed using an enhanced chemiluminescence detection reagent (WanleiBio, Shengyang, China). After sacrificing the mice and isolating the tumor tissue, Western blot was performed as previously described.