Immunoblotting was performed as described [14 (link)] with the following commercial primary antibodies: SMAD 1/5/9 (ab66737, Abcam, Cambridge, UK), total OXPHOS Antibody Cocktail (ab 110413, Abcam, Cambridge, UK), UCP1 (MAB6158, R&D System), β-tubulin (#2128), pSMAD 1/5/9 (#13820), Akt (#9272) and phospho-Akt (ser 473) (#9271) (all from Cell Signalling Technology, Danvers, MA, USA) and phospho-Akt (thr308) (#9275S BioLabs). Quantifications were performed by normalisation against loading controls.
Akt 9272
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
AKT (9272) is a primary antibody that recognizes AKT, a serine/threonine protein kinase that plays a central role in mediating the effects of growth factors on cellular processes such as metabolism, proliferation, cell survival, growth, and angiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
Pakt 4060, by Cell Signaling Technology (3 mentions)
Erk1 2 9102, by Cell Signaling Technology (2 mentions)
P akt 9271, by Cell Signaling Technology (2 mentions)
Ab97779, by Abcam (2 mentions)
Ab73734, by Abcam (2 mentions)
Ab182651, by Abcam (2 mentions)
Ecl kit, by Bio-Rad (2 mentions)
Foxo3a 2497, by Cell Signaling Technology (2 mentions)
Foxo1 2880, by Cell Signaling Technology (2 mentions)
Phospho akt ser473 9271, by Cell Signaling Technology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Total oxphos antibody cocktail, by Abcam (1 mentions)
Mab6158, by R&D Systems (1 mentions)
β tubulin, by R&D Systems (1 mentions)
P smad1 5 9, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Gapdh mab374, by Merck Group (1 mentions)
P erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
Loxo 292, by MedChemExpress (1 mentions)
Gfrα1, by R&D Systems (1 mentions)
33 protocols using akt 9272
1
Immunoblotting Antibody Quantification Protocol
2
Screening of RET Kinase Inhibitors
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Chemicals were from Sigma-Aldrich unless otherwise stated. Compound C3 (GW440139A), originally provided by GSK (UK), was re-synthesised at the ARUK Drug Discovery Institute at UCL [28 (link)]. LOXO-292 was purchased from MedChemExpress (USA). Recombinant human GDNF and BDNF were purchased from PeproTech (UK), and GFRα1 was purchased from R&D Systems (USA). RET shRNA constructs were purchased from Genecopoeia in psi-LVRU6GP plasmids with eGreen Fluorescent Protein (GFP) reporter genes and puromycin stable selection markers (MSH025822-LVRU6GP, USA). Antibodies used in immunofluorescence (IF) and western blot (WB) experiments were as follows: RET (C31B4, WB 1:1000, IF 1:250), RET pY905 (3221 1:1000), ERK1/2 (9102 1:1000), pERK1/2 T202/Y204 (9101 1:1000), AKT (9272 1:1000), pAKT S473 (4060 1:1000, Cell Signalling Technology (UK); GAPDH (mab374 1:5000, Merck Millipore, Germany); βIII-tubulin (IF 1:500, WB 1:3000, 302305, Synaptic Systems, Germany), GFP (GFP-1010, IF 1:1000, WB 1:5 000, Aves Labs, USA). The α-p75NTR used in the kinase inhibitor screen was previously generated and characterised by the authors [8 (link), 29 (link), 30 (link)].
3
Evaluation of SJDBT Extract in Cisplatin-Induced Toxicity
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Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and SJDBT was obtained from Hanpoong Pharm & Foods Co., Ltd., (Jeonju, Republic of Korea). SJDBT extract was prepared, according to the Good Manufacturing Practices guidelines (35 –37 ). Briefly, 10 of the components comprising SJDBT [Angelica gigas, Astragalus membranaceus, Atractylodes japonica, Cinnamomum cassia, Cnidium officinale, Paeonia lactiflora, Panax ginseng, Poria cocos and Rehmannia glutinosa (320 g each), and Glycyrrhiza uralensis (160 g)] were placed in 10-fold volume of water, boiled at 100°C for 3 h, filtered using 25 µm microfilters, and were subsequently extracted at 60°C. Finally, 876.9 g dried extract was obtained and the average yield was ~28.85%. Megestrol acetate (MA; Megace®) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Primary polyclonal antibodies against phosphorylated (p-)JAK1, (3331) p-STAT3 (9131), STAT3 (9132), p-AKT (9271) and AKT (9272) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-α-tubulin monoclonal antibody (T-5168) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
Investigating ACTL6A and FSHR in AKT Signaling
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ACTL6A (ab131272) and FSH receptor (FSHR) (ab75200) antibodies were from Abcam (Cambridge, MA, USA). ACTL6A (sc-137062) mouse monoclonal antibody was from Santa Cruz (Dallas, TX, USA). AKT (#9272) and Phospho-AKT (Ser473) (#4060) antibodies were from Cell Signaling Technology (Danvers, MA, USA). PGK1 (17811-1-AP), GAPDH (10494-1-AP), and secondary antibodies were from Proteintech (Wuhan, China). FSH (869001) was from Merck (Darmstadt, Germany). LY294002 (S1105) and MK2206 (S1078) were from Selleck (Houston, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) kit (C0009) was obtained from Beyotime Biotech, Inc. (Shanghai, China). Transwell® Polycarbonate Membrane (#3422) was obtained from Corning (NY, USA).
5
Western Blot Analysis of Rac1, ERK, and AKT
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SDS-PAGE (10%) was performed and Rac1 was detected using a rabbit polyclonal antibody #2465 (Cell Signaling Technology, Leiden, The Netherlands). GAPDH was used as a loading control #G9545 (Sigma-Aldrich, Taufkirchen, Germany). For analysis of ERK and AKT phosphorylation, cells were transfected using the TurboFect kit (Thermo Fisher Scientific, Rockford, Illinois, USA), left for 2 days in medium containing fibronectin-depleted FCS and harvested. The antibodies used were: pERK 1/2 (detecting phosphorylation at Thr202/Tyr185) #4376, ERK 1/2 #9102, pAKT (detecting phosphorylation at Ser473) #9271, AKT #9272 (All four antibodies from Cell Signaling Technology, Leiden, The Netherlands).
6
Mammary Tumor Protein Analysis
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Lysates were prepared from the mammary tumors of three Rarb-/- and
three wild-type mice. Antibodies against phospho-GSK3β (Ser9, 07-835), GSK3β
(07-1413), Snail (ABD38), phospho-AKT (9611), and AKT (9272) were purchased from Cell Signaling
Technology. Antibodies against RARβ (SC-552), Wnt-1 (SC-5630), Wisp1 (SC-13316), E-cadherin
(SC-7870), integrin α5 (SC-10719), LAMA1 (SC-56145), vimentin (SC-373717), cytokeratin
(SC-529), IGF1 (SC-7144), IGFBP5 (SC-6006), and actin (SC-1616) were purchased from Santa Cruz
Biotechnology.
three wild-type mice. Antibodies against phospho-GSK3β (Ser9, 07-835), GSK3β
(07-1413), Snail (ABD38), phospho-AKT (9611), and AKT (9272) were purchased from Cell Signaling
Technology. Antibodies against RARβ (SC-552), Wnt-1 (SC-5630), Wisp1 (SC-13316), E-cadherin
(SC-7870), integrin α5 (SC-10719), LAMA1 (SC-56145), vimentin (SC-373717), cytokeratin
(SC-529), IGF1 (SC-7144), IGFBP5 (SC-6006), and actin (SC-1616) were purchased from Santa Cruz
Biotechnology.
7
Kinase Inhibitor Library Evaluation
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The kinase inhibitor library (L1200), Trametinib, AZD8330 and TAK-733 inhibitors were purchased from SelleckChem (Huissen, Netherlands). The ERK (9102), phospho(44/42)-ERK (137F5), phospho(Ser2448)-mTOR (D9C2), phospho(Ser473)-AKT (#9271) and AKT (#9272) antibodies were from Cell Signaling (Bioké, Leiden, Netherlands). The antibody against tubulin (T-9026) was from Sigma Aldrich (Zwijndrecht, The Netherlands).
8
Western Blot Analysis of Protein Markers
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The proteins of interest were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a nitrocellulose membrane. Membranes were further blocked with 5% skim milk, and immersed into antibodies solution against ANXA9 (ab166621), MMP-2 (ab97779), MMP-9 (ab73734; all from Abcam), AKT (#9272), p-AKT (#9271), and GAPDH (#5174; all from Cell Signaling Technology, USA]. Then, the membranes were immersed into the secondary antibody solution linked to horseradish peroxidase (HRP; Beyotime, Shanghai, China). Signals were captured by a chemiluminescence system.
9
Western Blot Analysis of Inflammatory Signaling
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Cells were rinsed in PBS and scraped in 400 ml of cell lysis buffer containing protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). Cell lysates were then centrifuged at 12,000 rpm for 15 min at 4°C. The protein concentration in each lysate was then determined using the BCA assay. SDS-PAGE gels were loaded with the same amount of protein, and the NC membrane was blocked for 30 min with skimmed milk. Several primary antibodies were used for western blotting (as 1:1000 dilutions): NF-κB p65, ab16502; IkBα, ab76429; TLR2, ab108998; MyD88, ab219413; IRAK1, ab238; TRAF6, ab33915; TIRAP, ab17218; TAK1, ab109526; p-NF-κB p65, ab76302; IRAK4, 4363; p-PI3K, ab182651 (Abcam, Cambridge, MA, United States). We also used β-actin, 4970, GAPDH, 5174; AKT, 9272; and p-AKT, 4060S (Cell Signaling Technology, United States). HRP-labeled goat anti-rabbit secondary antibodies were used at a dilution of 1:2000 (A0208; Beyotime Biotechnology, Shanghai, China) and immunoreactive bands were detected using an Amersham Imager 600 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
10
Western Blot Analysis of AKT and FOXO1
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Cells were lysed with RIPA buffer (Beyotime, Shanghai, China). Equal amounts of proteins were separated on 8–12% SDS-PAGE gels according to different molecular weights, and then transferred onto polyvinylidene fluoride membranes (Millipore, Stafford, VA, USA). The membranes were then blocked with 5% bovine serum albumin, followed by incubation with primary and secondary antibodies. Proteins were visualized using an ECL kit (Bio-Rad, Hercules, CA, USA). Immunoblots were further subjected to semi-quantitative analysis using ImageJ software (National Institute of Health, Bethesda, MD, USA). The relative expression of target proteins was normalized to β-actin.
The following antibodies were used for immunoblots: FOXO1 (ab52857) and RAG1(ab172637) from Abcam (Boston, MA, USA); AKT (#9272), p-AKT (#9271), β-actin (#3700) as well as anti-mouse and anti-rabbit secondary antibodies from Cell Signaling (Danvers, MA, USA).
The following antibodies were used for immunoblots: FOXO1 (ab52857) and RAG1(ab172637) from Abcam (Boston, MA, USA); AKT (#9272), p-AKT (#9271), β-actin (#3700) as well as anti-mouse and anti-rabbit secondary antibodies from Cell Signaling (Danvers, MA, USA).
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