Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1)

Abstract

Generation of polyclonal antibodies specifi c to the ribosomal protein S6 kinase isoform-p85S6K1 and directed to the N-terminal (1-23 aa) extension of p85S6K1. Methods. Animal immunization with synthetic (1-23 aa) peptide, ELISA, western blot, immunoprecipitation, immunofl uorescent analysis. Results. Polyclonal antibodies have been generated, which specifi cally recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofl uorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension-the identifi cation of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms. K e y w o r d s: Ribosomal protein S6 kinase 1 (S6K1), S6K1 isoforms, p70 S6K1, p85 S6K1, polyclonal antibodies.

Generation and characterization of the MCF-7 cell line with a knockout of a p85-S6K1 isoform of the ribosomal protein S6 kinase 1

“…Proteins of interest were revealed using enhanced chemiluminescence reagent (GE Healthcare). Primary antibodies were used as follows: anti-S6K1-C-terminus (generated as described in [19]) 1:2500, anti-p85-S6K1-Nterminus (generated as described in [20] Immunofluorescence staining. All the cell lines used in this study were grown on the glass coverslips and fixed in 10 % neutral buffered formalin for 15 min.…”

“…Cells were then permeabilized using 0.2 % Triton X-100 and incubated in blocking solution (10 % fetal calf serum in PBS) for 30 min. After blocking, cells were treated with primary antibodies against the p85-S6K1 N-terminus [20] diluted 1:100 overnight at 4 °C. Coverslips were washed with PBS and then labeled with FITC-tagged secondary antibodies (Jackson Immuno-Research) diluted 1:400 for 1 h at room temperature.…”

Optimization of in vitro model for analysis of tumor cell migration dynamics

“…Non-specific antibody binding was blocked with 10 % FCS in PBS for 30 min at 37 °C. Anti-p85(S6K1) rabbit polyclonal antibodies were applied in dilution 1:100 overnight at 4 °C [18]. Secondary FITC conjugated anti-rabbit antibodies (Jackson Immuno Research Laboratories, Pennsylvania, USA) were applied in dilution 1:400 for 1h at 37 °C in humidified chamber.…”