Total protein was extracted from tissues and cell pellet lysis, and the concentration of protein in the lysate was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, United States). Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore; Merck KGaA, Darmstadt, Germany). After blocking with 5% skimmed milk for 1 h, the membranes were probed with primary antibodies at 4°C overnight: anti-TP53 (1:1000) and LC3 (1:500) were purchased from Cell Signaling Technology (CST; Danvers, MA, United States), while anti-p-AKTSer473 (1:500), anti-p-mTOR (1:500), anti-p-P70S6K, anti-p-4EBP1, and P62 (1:500) were purchased from Sigma (St. Louis, MO, United States), and anti-GAPDH (1:5000) was purchased from CST, which served as a loading control. Then, the membrane was incubated with a secondary antibody for 2 h. The protein expression was visualized using enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific), and the concentration of proteins was quantified by Image J software.
Anti p aktser473
Manufactured by Merck Group
Sourced in United States
Anti-p-AKTSer473 is a laboratory-grade antibody product designed for the detection and quantification of phosphorylated AKT at serine 473. This antibody is a key tool for researchers studying cell signaling pathways and the regulation of the AKT protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Merck Group (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti tp53, by Cell Signaling Technology (1 mentions)
Anti p p70s6k, by Merck Group (1 mentions)
Anti p mtor, by Merck Group (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Anti mbp, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti β actin, by MP Biomedicals (1 mentions)
Anti s6k, by Merck Group (1 mentions)
Anti s6, by Merck Group (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti tsc2, by Cell Signaling Technology (1 mentions)
Anti tsc1, by Cell Signaling Technology (1 mentions)
Anti myc, by Merck Group (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Hrp conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Anti bcl 2, by Merck Group (1 mentions)
5 protocols using anti p aktser473
1
Protein Quantification and Western Blot Analysis
2
Sciatic Nerve Protein Extraction and Analysis
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Sciatic nerves were homogenized on ice using lysis buffer for 5 min and then
centrifuged at 12,000×g for 15 min at 4°C. Afterwards, the protein concentration
of each supernatant was determined by ristocetin-induced platelet agglutination
(RIPA) of Tissue Protein Extraction Reagent (Beyotime, Shanghai, China). The
tissue extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and transferred to polyvinylidene difluoride membranes
(Millipore, Haverhill, MA). The membranes were incubated with the appropriate
primary antibodies overnight at 4°C: anti-Akt (1:1000; Sigma-Aldrich),
anti-p-Akt (ser-473) (1:1000; Sigma-Aldrich), anti-SIM312 (1:500;
Sigma-Aldrich), anti-MBP (1:500; Sigma-Aldrich), anti-β-actin (1:500;
Sigma-Aldrich), rabbit anti-GSK3B antibody (1:500; Abcam), rabbit polyclonal
anti-GSK3β antibody (1:500; Abcam, California, USA), and anti-GAPDH antibody
(1:1000; Sigma-Aldrich). Horseradish peroxidase–conjugated secondary antibody
was employed to visualize immunoreactivity using enhanced chemiluminescence; UVP
BioSpectrum Multispectral Imaging System (Ultra-Violet) was used for
densitometric analysis.
centrifuged at 12,000×g for 15 min at 4°C. Afterwards, the protein concentration
of each supernatant was determined by ristocetin-induced platelet agglutination
(RIPA) of Tissue Protein Extraction Reagent (Beyotime, Shanghai, China). The
tissue extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and transferred to polyvinylidene difluoride membranes
(Millipore, Haverhill, MA). The membranes were incubated with the appropriate
primary antibodies overnight at 4°C: anti-Akt (1:1000; Sigma-Aldrich),
anti-p-Akt (ser-473) (1:1000; Sigma-Aldrich), anti-SIM312 (1:500;
Sigma-Aldrich), anti-MBP (1:500; Sigma-Aldrich), anti-β-actin (1:500;
Sigma-Aldrich), rabbit anti-GSK3B antibody (1:500; Abcam), rabbit polyclonal
anti-GSK3β antibody (1:500; Abcam, California, USA), and anti-GAPDH antibody
(1:1000; Sigma-Aldrich). Horseradish peroxidase–conjugated secondary antibody
was employed to visualize immunoreactivity using enhanced chemiluminescence; UVP
BioSpectrum Multispectral Imaging System (Ultra-Violet) was used for
densitometric analysis.
3
Immunoblotting Detailed Protocol
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Immunoblotting was performed as described earlier (Zidek et al, 2015 ) with the following antibodies: from Cell Signaling Technology, anti‐TSC1 (#4906), anti‐TSC2 (#4308); anti‐pS6K (Thr389) (#9206) and (Thr389) (#9234), anti‐S6K (#9202), anti‐pS6 (Ser235/236) (#2211), anti‐S6 (#2317), anti‐pSAPK/JNK (Thr183/Tyr185) (#9251); anti‐p‐4E‐BP1 (T37/46) (#9459); anti‐p‐AKT (Ser473) (#4060); anti‐AKT (#9272); anti‐MYC (#5605); from Sigma‐Aldrich, anti‐β‐actin (A3853); from Santa Cruz Biotechnology, anti‐α‐tubulin (sc‐8035), anti‐TSC2 (sc‐893), anti‐MYC (sc‐40), anti‐SAPK/JNK (sc‐571); and from MP Biomedicals, anti‐β‐actin (#69100). Bands were visualized by chemoluminescence (Western Lightning Plus‐ECL, Perkin Elmer).
4
Protein Lysis and Western Blot Analysis
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Cells were lysed in ice-cold lysis buffer (20 mm Tris-HCl pH8, 300 mm KCl, 10% Glycerol, 0.25% Nonidet P-40, 0.5 mm EDTA, 0.5 mm EGTA, 1 × protease inhibitors) and lysates cleared by centrifugation (25 min, 15 000 × g, 4 °C) before PAGE and western blotting as previously described.10 (link) Primary antibodies used for western blot analysis were used at the dilution recommended by the manufacturer unless noted, and included a custom PRP4K antibody (H143, 1:1000);9 (link) antibodies from Cell Signaling (Danvers, MA, USA), anti-EGFR (#4267, 1:2000), anti-Akt (pan) (#4691, 1:2000), anti-p-Akt (Ser473) (#4060, 1:2000), anti-Erk (#4695), anti-pErk (#9106), anti-Bcl2 (#3498) and antibodies in the EMT sampler kit (#9782); Sigma, anti-actin (A2228) (1:5000); and Santa Cruz (Santa Cruz, CA, USA), anti-TrkB (sc-37721). Secondary antibodies used include HRP-conjugated goat anti-rabbit IgG (Sigma, A6154), HRP-conjugated sheep anti-mouse IgG (Sigma, A5906) and HRP-conjugated donkey anti-sheep IgG (Sigma, A3415).
5
Western Blot Analysis of Cell Signaling Proteins
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Western blot analysis was performed according to standard methods as previously described.27 , 28 Total protein was extracted from cell pellets or fresh tissue after harvest. The blots were immunostained with primary and secondary antibodies. Anti‐TEF (1:500) was purchased from Abcam (Cambridge, MA); anti‐Ki67 (1:1000) was purchased from Cell Signalling (Danvers, MA); and other antibodies, including anti‐p‐AKTSer473 (1:500), anti‐p‐AKTThr308 (1:500), anti‐AKT (1:500), anti‐CDK4 (1:500), anti‐CDK6 (1:500,), anti‐cyclin D1 (1:500), anti‐p21Cip1 (1:200), anti‐p27Kip1 (1:200), anti‐p‐FOXO1Ser256 (1:500), anti‐FOXO1 (1:1000), anti‐p‐FOXO4Ser197 (1:500) and anti‐FOXO4 (1:500), were purchased from Sigma (St. Louis, MO). The membranes were stripped and reblotted with an anti‐α‐tubulin monoclonal antibody (1:1000; Abcam), which served as a loading control.
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