Af1576

Protein aliquots of 25 μg each were separated by SDS polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were washed three times and then incubated with Blocking One solution (Nacalai Tesque, Inc., Kyoto, Japan) for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies against anti-RET (D3D8R) (#14698), anti-phospho-RET (Tyr905) (#3221), anti-AKT (#9272), anti-phospho-AKT (Ser473) (#4060), anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9664), and anti-β-actin (13E5) (#4970) antibodies (1:1,000 dilution each; Cell Signaling Technology, Danvers, MA). Additional antibodies were also used including anti-human/mouse/rat extracellular signal-regulated kinase (ERK) 1/ERK2 (0.2 μg/mL) (AF1576) and anti-phospho-ERK1/ERK2 (T202/Y204) (0.1 μg/mL) (AF1018) from R&D Systems. The membranes were washed three times and then incubated for 1 hour at room temperature with species-specific horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate, an enhanced chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). Each experiment was performed independently at least three times.

Total protein was extracted from CRC cells after 48h transfection. The Western blot assay was performed as previously described [34 (link)]. Immunoblotting was carried out with anti-PLEKHO2 (1:500, sc-100412, SANTA CRUZ, CA, USA), anti-ERK1/ERK2(1:1000, AF1576, R&D SYSTEMS, MN, USA), anti-pERK (1:500, AF1018, R&D SYSTEMS, MN, USA), anti-alpha Actinin1(1:400, AF8279, R&D SYSTEMS, MN, USA) and anti-c Myc (1:400, sc-40, SANTA CRUZ, CA, USA).

The animals received anesthesia (ketamine/200 μL/Kg and xylazine/400 μL/Kg) and diazepam (200 μL/Kg)) intraperitoneally. Then, insulin (200 mU) was administrated via i.c.v. by microinjection (stereotactic coordinates: anteroposterior −1.8 and depth −5.0) 5 min before the euthanasia. The procedures for the hypothalamus extraction and immunoblotting analysis have been previously described by our research group (Silva et al., 2014 (link)). The primary antibodies used were anti-Akt (rabbit, sc-8312, Santa Cruz Biotechnology^®), anti-phospho-Akt (Ser473; rabbit, sc-101629, Santa Cruz Biotechnology^®), anti-FoxO1 (rabbit, 9454s, Cell Signaling Technology^®), anti- β-Actin (rabbit, 8457, Cell Signaling Technology^®), Anti-phospho-Erk1/Erk2 (T202/Y204/T185/Y187; rabbit, AF1018, R&D Systems^®), anti-Erk1/2 (rabbit, AF1576, R&D Systems^®), anti-phospho-FoxO1 (Ser256; rabbit, 9461s, Cell Signaling Technology^®), anti-MKP-3 (rabbit, bs-11546R, Bioss^®). The luminescence of the samples present in the membranes was detected by a chemiluminescent reagent (ECL) and subsequently exposed to RX (Kodak^®) films and quantified by densitometry (UN-SCAN-IT gel 6.1 software).