Hrp conjugated goat anti mouse and anti rabbit secondary antibodies

The HTB11 neuroblastoma cell line were incubated overnight in EMEM medium containing low levels of BSA (0.5%) to render the cells quiescent. After the cells were stimulated with EPO (0.5 or 20 iU/ml), or the medium level with 10% FBS as a positive control at 37°C for 5 min, the cells were lysed for 10 min on ice in RIPA lysis buffer containing protease and phosphatase inhibitors (Santa Cruz Biotechnology). The extracted proteins were separated on a 4-12% SDS-PAGE gel and transferred to a PVDF membrane. Phosphorylation of the serine/threonine kinase AKT (yielding phospho-AKT⁴⁷³) and p44/42 mitogen-activated kinase (yielding phospho-^44/42 MAPK) was detected by phosphospecific p44/42 MAPK mouse and AKT rabbit polyclonal antibodies (Cell Signaling Technology, Danvers, MA, USA) with HRP-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Santa Cruz Biotechnology). Equal loading in the lanes was evaluated by stripping the blots and reprobing with anti-p42/44 MAPK monoclonal antibody (Cell Signaling Technology) and anti-AKT polyclonal antibody (Cell Signaling Technology). The membranes were developed with an enhanced chemiluminescence (ECL) reagent (Amersham Life Science, Arlington Heights, IL, USA), dried, and subsequently exposed to film (Hyperfilm; Amersham Life Science).

RMS cell lines were kept overnight in RPMI medium containing low levels of bovine serum albumin (BSA, 0.5%) to render the cells quiescent. After the cells were stimulated with LPA (0.1 μM) or LPC (20 μM) at 37°C for 5 min or 2 h, respectively, cells were lysed for 20 min on ice in RIPA lysis buffer containing protease and phosphatase inhibitors (Santa Cruz Biotech, Santa Cruz, CA). The extracted proteins were separated on a 12% SDS-PAGE gel and transferred to a PVDF membrane. The phosphorylation of the serine/threonine kinase AKT (phospho-AKT473) and p44/42 mitogen-activated kinase (phospho-p44/42 MAPK) was detected by phospho-specific p44/42 MAPK mouse and rabbit polyclonal antibodies (Cell Signaling, Danvers, MA, USA) with HRP-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Santa Cruz Biotech). Equal loading in the lanes was evaluated by stripping the blots and reprobing with anti-p42/44 MAPK monoclonal antibody (clone no. 9102, Cell Signaling) and anti-AKT polyclonal antibody (Cell Signaling). The membranes were developed with an enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences, Arlington Heights, IL), dried, and subsequently exposed to film (Hyperfilm, Amersham Life Sciences).