Anti phospho p44 42 map kinase thr202 tyr204

Purified phosphatidylcholine from soybean lecithin (Phospholipon 90G, CAS-number 97281-47-5) was purchased from Lipoid (Ludwigshafen, Germany). Trizma base, HEPES, Tween 20, Triton X-100, sodium dodecyl sulfate (SDS), glycine, ammonium persulfate, aprotinin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, 2-mercaptoethanol, Hoechst 33258, and BSA-fraction V were obtained from Sigma Chemical Co. (St. Louis, MO, USA). PVDF membranes, high performance chemiluminescence film, and enhanced chemiluminescence- (ECL-) Plus are from Amersham Biosciences (GE Healthcare, Piscataway, NY, USA). Mini-Protean apparatus for SDS-polyacrylamide electrophoresis, miniature transfer apparatus, acrylamide, bis-acrylamide, and TEMED were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Anti-EGFR (1005) antibody and secondary antibodies conjugated with HRP were purchased from Santa Cruz Biotechnology Laboratories (Santa Cruz, CA, USA). Antibodies anti-phospho-mTOR Ser2448, anti-mTOR, anti-p44/42 MAP kinase (ERK 1/2), and anti-phospho-p44/42 MAP kinase Thr202/Tyr204 were from Cell Signaling Technology Inc. (Beverly, MA, USA). Cy3-conjugated secondary antibody against rabbit polyclonal immunoglobulins was from Jackson ImmunoResearch Laboratories, Inc. Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific, Pierce Protein Research Products (Rockford, IL, USA).

Immunohistochemistry was performed as described in Papale et al. (2016 (link)). After quenching with 3% H₂O₂, 10% methanol for 15 min, the sections were rinsed in TBS and incubated for 1 h in blocking solution (5% normal goat serum, 0.1% Triton X-100). The sections were then incubated overnight at 4°C with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204; 1:1,000, Cell Signaling Technology, Danvers, MA, USA). On the following day, a biotinylated goat anti-rabbit secondary antibody (1:200, Vector Labs) was applied to the sections for 2 h at room temperature. Detection of the bound antibodies was carried out using a standard peroxidase-based method (ABC-kit, Vectastain, Vector Labs), followed by incubation with DAB and H₂O₂ solution. The sections were subsequently dehydrated using increasing concentrations of ethanol and mounted with DPX. Images were acquired from the striatum using a bright-field microscope (Leica DMI6000B Macro/Microimaging system) under a 20× magnification.