The antibodies against TC-PTP (TC45, #58935), pSTAT3 (Y705) (#9145), STAT3 (#9139), pAKT (S473) (#4060), AKT (#4685), Phospho-AKT substrate (#9614), Bcl-xL (#2764), Bax (#2772), Cleaved Caspase 3 (#9661), Cleaved PARP (#9548), Cyclin D1 (2978) and α/β tubulin (#2148) were purchased from Cell Signaling Technology. The antibodies against Lamin A/C (sc-7293), c-Myc (sc-40), β-actin (sc-47778), and LDH (sc-33781) were bought from Santa Cruz Biotechnology. The antibody against 14-3-3σ (GTX100801) was purchased from GeneTex. Anti-V5 tag antibody (R960-25) was purchased from Invitrogen and used for detection of exogenous TC45. STA-21, S3I-201, AKT1/2 inhibitor, Staurosporine, Gö6983, Ro-31-8220, Gö6976, spermidine, protease inhibitor cocktail, phosphatase inhibitor cocktail I, phosphatase inhibitor cocktail II and Leptomycin B were purchased from Sigma-Aldrich.
Akt1 2 inhibitor
Manufactured by Merck Group
Sourced in United States
AKT1/2 inhibitor is a laboratory reagent used in cell-based assays and biochemical studies. It functions as a selective inhibitor of the AKT1 and AKT2 protein kinases, which are involved in cellular processes such as cell growth, proliferation, and survival. The product can be used to investigate the role of the AKT signaling pathway in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail 2, by Merck Group (2 mentions)
Staurosporine, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail 1, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Ro31 8220, by Merck Group (1 mentions)
Spermidine, by Merck Group (1 mentions)
G 6976, by Merck Group (1 mentions)
Leptomycin b, by Merck Group (1 mentions)
Sta 21, by Merck Group (1 mentions)
Ldh sc 33781, by Santa Cruz Biotechnology (1 mentions)
Anti v5 tag antibody r960 25, by Thermo Fisher Scientific (1 mentions)
G 6983, by Merck Group (1 mentions)
α β tubulin, by Cell Signaling Technology (1 mentions)
Gsk1016790a, by Merck Group (1 mentions)
Rn 1734, by Merck Group (1 mentions)
12 protocols using akt1 2 inhibitor
1
TC-PTP Signaling Pathway Analysis
2
Modulation of Megakaryocyte Collagen Adhesion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human CD34+ cells were isolated, separated and cultured, as described previously.27 (link),28 (link) All human samples were collected in accordance with the ethical committee of the IRCCS Policlinico San Matteo Foundation, Pavia, Italy, and the principles of the Declaration of Helsinki. For collagen receptor inhibition, MKs at day 13 of culture were incubated with 10 μg/mL anti-β1 integrin blocking antibody (Millipore, clone P5D2), 10 μg/mL anti-GPVI blocking antibody (a kind gift of Prof. Jandrot Perrus) or with 200 nM (125 ng/mL) Discoidin Domain Receptor 1 (DDR1)-IN-1 (Tocris), a selective DDR1 tyrosine kinase inhibitor, for one hour prior to being plated on type I or type IV collagen for three hours (h). For Akt inhibition experiments, MKs at day 13 of culture were treated with 10 μM Akt1/2 inhibitor (Sigma Aldrich) for 30 minutes (min) and then plated on collagens for 16 h for PPT evaluation. For treatment with the TRPV4 inhibitors (RN-1734, HC067047, Sigma Aldrich), MKs at day 13 of culture were incubated with vehicle or 10 μM of the indicated TRPV4 inhibitor for 30 min prior to being plated on collagens for 3 or 16 h. For treatment with the TRPV4 agonist (GSK1016790A, Sigma Aldrich), MKs at day 13 of culture were incubated or not with 10 μM GSK1016790A for 10 min prior to being plated on collagens for 3 h.
3
Investigating IGF-I/II Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IGF-I and IGF-II were from Preprotech (Rocky Hill, NJ), lipofectamine 2000 and lipofectamine RNAiMax from Life Technologies Inc. Laboratories (Paisley, UK), scramble (miR-NC), synthetic pre-miR-199a-5p (miR-199a-5p), and mirVana miR-199a-5p inhibitor were purchased from Ambion (Applied Biosystems, CA, USA), fibronectin from Sigma-Aldrich (Saint Louis Missouri, USA).
Constructs encoding either pcDNA.3.1-HA-myr-AKT dominant active construct (AKT) or the empty vector pcDNA3.1 (vector) were kindly provided by prof. P. Tassone (University “Magna Graecia” of Catanzaro).
Construct encoding 3′UTR clone of DDR1 in pMirTarget Vector, the related empty vector (pCMV6) and the human IGF-II cDNAs were from OriGene Technologies (Rockville, USA). The following kinase inhibitors were used: the IGF-IR inhibitor NVP-AEW541 (Cayman Chemical, Ann Arbor, USA); the PI3 kinase inhibitor LY 294002 (Calbiochem, Merck Millipore, Nottingham, UK); the MEK1 inhibitor U0126 (Sigma-Aldrich, Saint Louis Missouri, USA); the TORC1 inhibitor rapamycin (Sigma-Aldrich); the AKT 1,2 inhibitor (Sigma-Aldrich, Saint Louis Missouri, USA). Actinomycin D and cycloheximide were purchased from Sigma-Aldrich, MTT from Amersham Bioscences.
Constructs encoding either pcDNA.3.1-HA-myr-AKT dominant active construct (AKT) or the empty vector pcDNA3.1 (vector) were kindly provided by prof. P. Tassone (University “Magna Graecia” of Catanzaro).
Construct encoding 3′UTR clone of DDR1 in pMirTarget Vector, the related empty vector (pCMV6) and the human IGF-II cDNAs were from OriGene Technologies (Rockville, USA). The following kinase inhibitors were used: the IGF-IR inhibitor NVP-AEW541 (Cayman Chemical, Ann Arbor, USA); the PI3 kinase inhibitor LY 294002 (Calbiochem, Merck Millipore, Nottingham, UK); the MEK1 inhibitor U0126 (Sigma-Aldrich, Saint Louis Missouri, USA); the TORC1 inhibitor rapamycin (Sigma-Aldrich); the AKT 1,2 inhibitor (Sigma-Aldrich, Saint Louis Missouri, USA). Actinomycin D and cycloheximide were purchased from Sigma-Aldrich, MTT from Amersham Bioscences.
4
Signaling Pathways in Cell Lines
5
Transwell Invasion Assay with AKT Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell chambers (8-µm pore size; BD Falcon, Franklin Lakes, NJ) were coated with the appropriate amount of 30 µg Matrigel (BD Biosciences, San Jose, CA), and 2.5×104 cells were suspended in 10% NuSerum-containing media (Gibco BRL, Grand Island, NY, USA), seeded in the chamber, and cultured for 20 hours (CL1-5 and A549) or 24 hours (BEAS-2B and CL1-1). To prevent the activation of AKT, 1×105 cells were pre-treated with LY294002 (50 µM for the BEAS-2B cells) or AKT1/2 inhibitor (7.5 µM for the CL1-5 cells) (Sigma-Aldrich, St. Louis, MO) for 24 hours, and then cells were seeded in the chamber with LY294002- or AKT inhibitor-containing media. Cells that invaded the chamber from top to bottom were fixed with methanol and stained with a 50 µg/mL solution of propidium iodide (Sigma-Aldrich, St. Louis, MO). The propidium iodide-positive signal was quantified using the Analytical Imaging Station software package. Each sample was assayed in triplicate.
6
High-Throughput Drug Screening in Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines were plated at 2000 cells per well in 384 well plates for 24 h at 37 °C. Detailed information on the cell lines and their growth conditions is provided in Additional file 2 : Sheet 1. All cell lines were obtained from American Type Culture Collection (ATCC). Drugs were diluted to six doses in media containing 5% FBS (Gibco/Life technologies) and 1% anti–anti (Gibco/Life technologies). Erlotinib, trametinib, UMI-77, obatoclax, doxorubicin, and neratinib were purchased from Selleckchem, and bafilomycin and AKT1/2 inhibitor were from Sigma-Aldrich. Drugs were dissolved in 100% DMSO and stored at −80 °C. Detailed information on drug doses is provided in Additional file 2 : Sheet 2. Cell viability and growth was measured using CellTiter-Glo (Promega) 72 h post-treatment. All treatment doses were performed in four replicates. The Drug Discovery Core Facility, a part of the Health Sciences Cores at the University of Utah, performed the dose response assay. EC50s (concentration of each drug that provides half of the maximum response) were determined and converted to drug sensitivity values defined as the negative log of the EC50s (−logEC50) (Additional file 2 : Sheet 3). EC50 values were calculated from dose response data by plotting in GraphPad Prism 4 and using the equation Y = 1/(1 + 10ˆ((logEC50 − X) × HillSlope)) with a variable slope (Ymin = 0 and Ymax = 1).
7
Regulation of ATF4 and Rictor in Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The eIF2αP-proficient or -deficient MEFs, HT1080 and A549 tumor cells were generated as described previously.30 (link) ATF4 KO MEFs as well as PERK/GCN2 KO MEFs were previously described.41 (link), 59 (link) Cells were cultured in Dulbecco modified Eagle medium (Wisent, St-Bruno, QC, Canada) supplemented with 10% fetal bovine serum (FBS; Gibco, Burlington, ON, Canada), antibiotics (100 U/ml of penicillin–streptomycin; Gibco) and 2.5 μg/ml of puromycin (Sigma, Oakville, ON, Canada). The shRNA-mediated KO of ATF4 in HT1080 cells was carried out based on previously reported protocol.69 (link) Lentiviral shRNA targeting ATF4 (TRCN0000013573) was obtained from the RNAi Consortium (TRC) arrayed human genome-wide shRNA collection (Sigma). shRNA-mediated KO of Rictor was performed as described previously.56 (link) H2O2 was purchased from Bioshop, Canada; GDC-0941 was obtained from Selleckchem, USA; thapsigargin, rapamycin, Akt1,2 inhibitor, PAO, PEITC and erastin were obtained from Sigma.
8
Insulin-Signaling Pathway Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Insulin solution [10 mg/mL], AKT1/2 inhibitor, cycloheximide, actinomycin D, protease inhibitor cocktail, phosphatase inhibitor cocktail I and phosphatase inhibitor cocktail II were purchased from Sigma-Aldrich (St. Louis, MO). Insulin was diluted in basal medium to the necessary concentrations for experimentation as needed. Polybrene [10 mg/mL] was purchased from Millipore (St. Louis, MO) and diluted in medium as needed. Anti-ADH1B, anti-ADH1C, anti-FABP4, and anti-adiponectin antibodies were purchased from Abcam Inc. (Cambridge, MA). Anti-ADH1A was purchased from Origene Technologies, Inc. (Rockville, MD). Anti-DDK, anti-Akt, anti-Phospho-Akt, and anti-PPARγ were purchased from Cell Signaling (Danvers, MA). Anti-GLUT4 was purchased form Bioss, Inc (Boston, MA). Anti-β-actin and IRDye® secondary antibodies were purchased from LI-COR (Lincoln, NE). All reagents were prepared and stored according to the manufacturer’s instructions.
9
Isolation and Characterization of Cytoskeletal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ficoll-Paque was obtained from Pharmacia (Uppsala, Sweden). Fibronectin was from Calbiochem (La Jolla, San Diego, CA, USA). Bicarbonate-free Hank’s solution, Ca2+-free Dulbecco PBS, cytochalasin D, minoxidil, doxycycline, wortmannin, Akt 1/2 inhibitor, cytochalasin D, latrunculin A, blebbistatin, staurosporine, 4-bromophenacyl bromide, and E64 were obtained from Sigma (Steinheim, Germany). Analytical chromatography conditions: eluent MCI Buffer L-8800-PH-1–4 and ninhydrin coloring solution kit for Hitachi 29,970,501 (Wako Chemicals, North Chesterfield, VA, USA). Coomassie Brilliant Blue G-250 was obtained from Serva, PMSF from MP Biomedical, trypan blue from Fluka AG, trypsin from Promega, glutaraldehyde from Ted Pella, carboxy-H2DCF-DA from Molecular Probe, (Eugene, OR, USA).
10
Silencing ANGPTL3 in Immortalized Hepatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver IHHs immortalized by SV40 large T-Antigen (IHH, ATCC® PTA-5565™) were transduced with MISSION™ shRNA (small hairpin RNA) Lentiviral Vector particles (TRCN0000242782, Sigma Aldrich) targeting ANGPTL3 (NM_014495.2) or with non-target shRNA (SHCOO2, Sigma Aldrich) [MOI (multiplicity of infection) 1]. Positive cells were selected against 5 μg/ml puromycin for 12 days. The transduction was repeated once after the first selection. Cells were cultured in Williams medium E (Gibco by Life Technologies, 22551-022) with added 10% (v/v) FBS and glutamine 0.2 mg/ml and incubated +37 °C. FBS was removed during experiments and total protein from cell lysates was used to normalize the data. Cells were washed with PBS (pH 7.4) and lysed in RIPA buffer. Protein concentration was measured with Bradford protein assay (Bio-Rad). Following compounds were used: insulin (bovine, Sigma-Aldrich), Wortmannin (Sigma-Aldrich), Akt1/2 inhibitor (Sigma-Aldrich), rosiglitazone (Cayman Chemical) and GW9662 (Sigma-Aldrich).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!