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P70 S6 kinase alpha is a Serine/Threonine-protein kinase that is upregulated by mTOR (in response to growth factors). The Ribosomal Protein S6, Eukaryotic Elongation Initiation Factor 4B (EIF4B) and Eukaryotic Elongation Factor-2 Kinase (EEF2K) are phosphorylated by p70 S6 kinase to promote cell survival, whilst p70 S6 kinase also represses the pro-apoptotic function of Bcl-2-associated death promoter (BAD).
1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: Phospho-p70 S6 kinase alpha (S447) Polyclonal Antibody (STJ90374)
2. WHAT WAS USED? Primary Antibody: Phospho-p70 S6 kinase alpha (S447) Polyclonal Antibody (STJ90374) Provider: St John’s Laboratory Dilution ratio: 1:500 Application: Western Blot Materials for validation: A549 & NIH3T3 cells
3. WHAT WAS THE PROTOCOL? Treatment of materials: Cells were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 1% SDS, supplemented with protease inhibitors (PMSF 1mM, Leupeptin 1ug/ml, Aprotinin 1ug/ml, Pepstatin 1ug/ml). Proteins were reduced and denatured by heating at 100¡æ for 5 min with subsequent cooling before use. Gel electrophoresis: 60ug/lane Transfer information: Nitocellulose membrane. Blocking: Blocked with 5% BSA for 0.5-1hr Primary Ab Application: Overnight at 4oC, 5% BSA in TBST. Secondary Ab Application: After washing with TBST 3 times membrane incubated secondary antibody for 1h at room temperature. Visualization: ECL kit
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