Equal volumes of total protein lysates were separated on a denaturing polyacrylamide gel, which were then processed for wet transfer onto a nitrocellulose membrane followed by probing of the blots with antibodies to detect phospho-p70 S6 kinase (Thr421/Ser424), phospho-Akt (Ser473), phospho-mTOR (Ser2448), phospho-PKC, total p70 S6 kinase, total Akt, total mTOR, and LC3-II (Cell Signaling Technology) and a compatible horseradish peroxidase (HRP)-conjugated secondary antibody for enhanced chemiluminescence-based detection. To normalize for variations in the protein loading on individual lanes, the blots were stripped and re-probed with an antibody against α-tubulin (Accurate Chemical and Scientific Corporation, Westbury, NY, USA).
Total p70 s6 kinase
Manufactured by Cell Signaling Technology
Sourced in United States
Total p70 S6 kinase is a quantitative sandwich ELISA kit designed for the measurement of total p70 S6 kinase levels in cell and tissue lysates.
Automatically generated - may contain errors
Lab products found in correlation
Phospho p70 s6 kinase, by Cell Signaling Technology (4 mentions)
Total mtor, by Cell Signaling Technology (4 mentions)
Total 4e bp1, by Cell Signaling Technology (3 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Phospho mtor ser2448, by Cell Signaling Technology (2 mentions)
Lc3 2, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Phospho pkc, by Cell Signaling Technology (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Odyssey sa infrared imaging system, by LI COR (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
0.5 m edta solution, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Rictor, by Cell Signaling Technology (1 mentions)
Raptor, by Cell Signaling Technology (1 mentions)
8 protocols using total p70 s6 kinase
1
Immunoblotting Analysis of Signaling Proteins
2
Western Blot Analysis of Phosphorylated Proteins
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Cells were lysed in Pierce RIPA buffer (Thermo Scientific) with the addition of 0.5M EDTA solution (Thermo Scientific) and Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific) according to the manufacturer’s instructions. Protein concentration was assessed by Bradford protein assay (Bio-Rad). Protein was separated at the stated concentrations by SDS-PAGE. 4–15% Mini- PROTEAN TGX gels were used (Bio-Rad), and the membrane was blocked in Fluorescent Blocker (Merck Millipore) for 1 hour at room temperature. Primary antibodies were applied overnight at 4°C at a concentration of 1:1000 for phospho-p70S6 Kinase, 1:1000 total-p70S6 Kinase and 1:2000 for β-actin (Cell Signal Technology, Inc). Washed membranes were incubated for 1 hour at room temperature with secondary antibodies at a concentration of 1:10000. A final series of washes were then performed before scanning the membranes (Odyssey SA Infrared Imaging System; LI-COR Bioscience). Blots were quantified using ImageJ software. For normalisation phosphorylated proteins were quantified against their corresponding total abundance to verify relative amount of analysed proteins.
3
Western Blot Analysis of Signaling Proteins
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Total protein lysate samples were analyzed on 8, 10, or 15% polyacrylamide gel based on the protein size and transferred onto a PVDF membrane (BioRad, Hercules, CA, USA), followed by probing the blots with primary antibodies and then horseradish peroxidase (HRP)-linked secondary antibodies for chemiluminescence-based detection. We used phospho-p70 S6 kinase (Thr412/Ser424, catalog #9204), phospho-Akt (Ser473, catalog #4058), phospho-mTOR (Ser2448, catalog #2971), total p70 S6 kinase (catalog #9202), total Akt (catalog #4691), total mTOR (catalog #2983), LC3II (catalog #2775), Raptor (catalog #2280), and Rictor (catalog #9476) protein expression antibodies from Cell Signaling Technology. The blots were stripped and reprobed with an α-tubulin antibody to account for variations in the protein loading.
4
CLL Cell Protein Expression Analysis
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Cells were untreated or treated with DMSO or AZD1208. CLL cells were harvested after treatment, centrifuged, and washed twice with PBS. Cells were lysed using one tablet of Complete Mini Protease Inhibitor Cocktail (Roche) in 10 mL of 1 x radioimmunoprecipitation assay buffer (Bio-Rad; Hercules, CA, USA) or 1 x Lysis Buffer (Cell Signaling). The lysate protein content was measured using a DC protein assay kit (Bio-Rad), according to the manufacturer’s instructions. Protein samples were electrophoresed on Criterion XT Bis-Tris gels using either XT MOPS buffer or XT MES buffer (Bio-Rad) and were transferred either to nitrocellulose or PVDF membranes. Primary antibodies were purchased from the following sources: BCL-2 (Dako; Carpinteria, CA, USA); MCL-1, BCL-XL, and c-MYC (clone C33) (Santa Cruz Biotechnology; Santa Cruz, CA, USA); phospho-4E-binding protein 1 (4E-BP1) (Thr37/46), phospho-4E-BP1 (Ser65), total 4E-BP1, phospho-p70 S6 kinase (Thr389), total p70 S6 kinase, glyceraldehyde 3-phosphate dehydrogenase, total Bad, total histone H3, and LC3A/B (Cell Signaling Technology; Danvers, MA, USA); phospho c-MYC-1 (Abcam; Cambridge, MA, USA); phospho-histone H3 (Ser 10) (EMD Millipore; Billerica, MA, USA); and poly ADP ribose polymerase (BD Pharmingen); p62 (Enzo Life Sciences, Farmingdale, NY).
5
Cardiac Protein Fractionation and Immunodetection
6
Investigating Quercetin and TFQ Effects
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Quercetin was purchased from Aladdin chemistry Co. Ltd whereas TFQ was synthesized at our laboratory. Each compound was dissolved in DMSO to prepare a stock solution of 50 mM. Antibodies for the protein characterization were against total p70S6 kinase, phosphor-p70S6 kinase (Thr389), total AMPK, phosphor-AMPK (Thr172), phosphor-mTOR (S2448), total –mTOR, total 4E-BP1, phosphor-4EBP1(Thr-70) and β-actin were purchased from Cell Signaling Technology. Compound C was from Selleckchem (Houston, Texas, USA).
7
Western Blot Analysis of Signaling Pathways
8
Bayberry Extract Modulates Autophagy
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Chinese bayberry crude extracts (CCE) were prepared as previously described [17 (link)]. Isoquercitrin and anti-LC3B antibody were purchased from Sigma-Aldrich (MO, USA). Antibodies against phospho-Ser2481-mTOR (Cat. 2974), mTOR (Cat. 2983), AMPKα (Cat. 5831), phospho-Thr172-AMPKα (Cat. 2535), cleaved-PARP (Cat. 5625), cleaved-caspase-3 (Cat. 9664), p62 (Cat. 8025), Beclin-1 (Cat. 3495), Atg5 (Cat. 2630), phospho-Thr389-p70S6 kinase (Cat. 9234), total p70S6 kinase (Cat. 9202), and GAPDH (Cat. 2118) were purchased from Cell Signaling Technology (MA, USA). Antibodies against Bax (Cat. ab32503) and Bcl-2 (Cat. ab182858) were obtained from Abcam (Cambridge, UK). 3-Methyladenine (3-MA), Bafilomycin A1 (Baf A1), Rapamycin (RAPA), z-VAD-FMK, and dorsomorphin were purchased from MCE (NJ, USA). Alexa Fluor 488 anti-rabbit IgG was bought from Invitrogen. Other chemicals and reagents were obtained from Sigma-Aldrich.
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