Alkaline phosphatase linked anti rabbit igg

Cell extracts of C6 cells grown in 35-mm culture dishes were prepared as previously described [40 (link)]. Equal amounts of proteins (20 μg for pCREB and 10 μg for others) were separated on SDS-polyacrylamide gels and electroblotted onto an Immuno-Blot™ PVDF membrane (Bio-Rad Lab., Hercules, CA, USA). As primary antibodies, rabbit polyclonal antibodies against 44/42 ERK1/2 and phospho-44/42 MAPK (Thr202/Tyr204) were purchased from Cell Signaling Tech. Inc. (Woburn, MA, USA). Rabbit polyclonal antibodies against actin and GDNF were purchased from Sigma-Aldrich Company Ltd. (St. Louis, MO, USA) and Abcam plc. (Cambridge, Cambridgeshire, UK), respectively. As secondary antibody, alkaline phosphatase-linked anti-rabbit IgG (Cell Signaling) were used. Immunoreactive bands were detected by use of the NCB/BCIP reagent (Roche Diagnotics GmbH, Mannheim, Germany).

Neuro2a cells were seeded in wells of a 6-well plate at a density of 2.5 × 10⁵ cells/well. The cell extracts were prepared as previously described [39 ]. Equal amounts of proteins (20 μg) were separated on SDS-polyacrylamide gels and electroblotted onto an Immuno-Blot^TM PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). As primary antibodies, rabbit polyclonal antibodies against 44/42 ERK1/2, which recognize 44-kDa ERK1 and 42-kDa ERK2; phospho-44/42 MAPK (Thr202/Tyr204), specific for phosphorylated ERK1/2 (pERK1/2); Akt; and phosphor-Akt (Ser473), which recognize phosphorylated Akt (pAkt), were purchased from Cell Signaling Technology Inc. (Woburn, MA, USA). As secondary antibody, alkaline phosphatase-linked anti-rabbit IgG (Cell Signaling) was used. Immunoreactive bands were detected by use of the NCB/BCIP reagent (Roche Diagnotics GmbH, Mannheim, Germany).