Anti erk2

After centrifugation and supernatant discard, the proteins were extracted from the pellet using RIPA Buffer and quantified using Bradford Assay (Bio-Rad, Hercules, CA, USA). After denaturation at 95 °C, 25 µg of protein for each samples underwent separation using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then they were transferred on PVDF membrane (Immobilon–P, Millipore, Burlington, MA, USA). Then, 5% skimmed milk in TBS was used for blocking at room temperature for 1 hour, followed by the overnight incubation at 4 °C. The antibodies used were: Anti-CB1 (1:500, ThermoFisher Scientific, Rockford, IL, USA), anti-GAPDH HRP conjugate (1:1000, Cell Signaling, Danvers, MA, USA), anti-p-Akt (1:1000; Cell Signaling, Danvers, MA, USA), anti-Akt (1:1000, Cell Signaling, Danvers, MA, USA), anti-p-ERK1/2 (1:2000; Cell Signaling, Danvers, MA, USA) and anti-ERK2 (1:1000; Cell Signaling, Danvers, MA, USA). The antibody mouse anti-rabbit IgG-HRP (1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a secondary antibody to be incubated with the membranes for 1 h at room temperature. In order to acquire the bands, ChemiDoc™ MP System (Bio-Rad) was after exposition to enhanced chemiluminescence system (Luminata Western HRP Substrates, Millipore, Burlington, MA, USA).

Paraffin-embedded sections were deparaffinized and immersed in unmasking solution (Vector H3300; Vector Laboratories) for antigenic retrieval and heated in an autoclave (121 °C) for 5 min. Sections were then incubated with a non-specific blocking reagent (Dako) for 1 h to block any non-specific reactions, followed by overnight incubation at 4 °C with one of the following primary antibodies: anti-ERK1 (Invitrogen, 13–8600), anti-ERK2 (Cell Signaling, #9108), anti-p-ERK1/2 (Cell Signaling, #9101), anti-myosin VIIa (Proteus Biosciences, 25–6790), or anti-CtBP2 (BD Transductions, 612044) diluted in an antibody diluent (DAKO). Sections were washed thrice in PBS and incubated with a corresponding secondary antibody (Alexa Fluor 488 or 546, IgG, Invitrogen) diluted in an antibody diluent (DAKO). After rinsing in PBS, NSE was mounted onto slides with an antifade mounting medium (VECTASHIELD with DAPI; Vector Laboratories, Burlingame, CA, USA). DAPI labeling was then used to identify condensed HC nuclei. Images of immunolabeled specimens were obtained by confocal fluorescence microscopy using a Nikon C2 system (Nikon, Tokyo, Japan).