Rabbit anti p erk 4370

Stainings were performed on FFPE archival patient tumor material by our in-house NKI-AVL Core Facility Molecular Pathology & Biobanking (CFMPB) for hematoxylin–eosin, rabbit anti-p-ERK (4370, Cell Signaling), and mouse anti-BRAF^V600E (VE1, Spring Bioscience). Xenografted tumors were stained by our in-house Animal Pathology Facility for Sirius Red and hematoxylin–eosin.

The spinal cord of the lumbar (L₄-L₅) ipsilateral quadrant to the lesion was collected, dissected, and homogenized in protein lysis buffer in the presence of protease inhibitors and incubated on ice for 10 min. The samples were centrifuged at 12,000 rpm for 15 min at 4°C. The total protein content was determined in the supernatants using the Bio-Rad DC Protein Assay Kit. Equal amounts of protein were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature and incubated overnight at 4°C with primary antibodies (rabbit anti-Iba1, ab178680, 1 : 1000, Abcam, USA; rabbit anti-BDNF, ab108319, 1 : 1000, Abcam, USA; rabbit anti-p-ERK, #4370, 1 : 1000, Cell Signaling Technology, USA; rabbit anti-PI3K, ab40776, 1 : 2000, Abcam, USA). Then, the membranes were incubated with a horse-radish peroxidase-conjugated secondary antibody (1 : 5000, Thermo Scientific, USA) at room temperature for 2 h. Finally, peroxidase activity was visualized using the ECL Western Blot Detection Kit (Beyotime, China). Western blots were quantitated using an image analysis system (Bio-Rad, USA). After normalization with β-actin, the data were presented as mean percentages of the ratio of total protein to their respective signal intensity levels found in the Sham group animals, indicated as 100%.

Cells seeded on coverslips were fixed for 15 min in 1.6% PFA at RT and permeabilized either with 100% ice cold methanol or 0.05% Saponin solution. Cells were stained with primary antibodies (rabbit anti-pERK, #4370, 1:200 or rabbit anti-pS6, #2215, 1:200; both Cell Signaling Technology) in combination with goat anti-GFP (ab5450, 1:2000, Abcam (Cambridge, UK)) overnight at 4°C, followed by 1 hour staining with secondary antibodies (donkey anti-rabbit DL549 and donkey anti-goat Alexa 488) at RT and counterstained with DAPI (all from Life Technologies). Fluorescence images were taken using Zeiss LSM confocal microscope (Zeiss, Oberkochen, Germany) or inverse microscope Olympus IX8 (Olympus, Norway) and analyzed with ZEN 2009 Light Edition or Cell^P softwares, respectively.
Sections of paraffin embedded tumor tissue were deparaffinized with xylene and ethanol and boiled in antigen unmasking solution (1:100, Vector laboratories Inc, Burlingame, CA) for 15 min in a microwave. The slides were stained as above, except that anti-GFP was diluted 1:1000.