Cells were collected and lysed. The protein contents were determined. Samples were resolved by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. Afterward, membranes were incubated respectively with an anti-Belcin1(1:1000 dilution, Abcam, USA), anti-LC3(1:1000 dilution, Abcam, USA), anti-phosphorylated PI3K (p-PI3K, 1:1000 dilution, Abcam, USA) and total PI3K (t-PI3K, 1:1000 dilution, Abcam, USA), anti-phosphorylated Akt (p-Akt, 1:1000 dilution, Millipore, USA) and total Akt (t-Akt, 1:1000 dilution, Millipore, USA), anti-mTOR (1:1000 dilution, Millipore, USA) and anti-GAPDH antibodies 1:1000 dilution, Millipore, USA) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG for 1 h at room temperature. The signals were enhanced using a chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, England).
P akt
Manufactured by Merck Group
Sourced in United States, United Kingdom, Denmark
P-Akt is a laboratory equipment product manufactured by Merck Group. It is used for the detection and quantification of phosphorylated Akt (Protein Kinase B), a key signaling protein involved in various cellular processes. The core function of P-Akt is to provide researchers with a reliable and accurate tool to measure the activation state of Akt in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (7 mentions)
P erk1 2, by Merck Group (4 mentions)
P erk, by Merck Group (4 mentions)
P jnk, by Merck Group (3 mentions)
P mtor, by Merck Group (3 mentions)
Vinculin, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Bcl2 associated x (bax), by Merck Group (2 mentions)
Bcl 2, by Merck Group (2 mentions)
Caspase 3, by Merck Group (2 mentions)
Gapdh, by Merck Group (2 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
T akt, by Merck Group (2 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Chemiluminescence system, by Cytiva (1 mentions)
Anti mtor, by Merck Group (1 mentions)
T pi3k, by Abcam (1 mentions)
Anti lc3, by Abcam (1 mentions)
31 protocols using p akt
1
Western Blot Analysis of Autophagy Markers
2
Multiplex Analysis of Insulin and Cytokine Signaling
3
Antibody Sourcing for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HA and Ran antibodies were obtained from Abcam (ab18181, ab157213). Kapβ2 antibody was obtained from Santa Cruz Biotechnology (sc-11368). GAPDH and histone antibody were purchased from Cell signaling Technology (2118, 8135). PTEN, p-AKT and total AKT antibodies were purchased from Millipore, USA (04-035, 05-1003, 16-294).
4
Protein Signaling Pathway Analysis
5
ONC201 Preclinical Evaluation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ONC201 was utilized from Oncoceutics for in vitro and in vivo studies. ONC201 was dissolved in dimethylsulfoxide (DMSO) at 10 mM stock concentration and diluted freshly to working concentrations for all in vitro studies. For in vivo experiments, the desired dose was prepared by dissolving the drug in olive oil (diluent) for oral administration (PO) via gavage. Antibodies were purchased from Cell Signaling Technology, and Millipore Sigma; PARP (#9542), Bim (#2933), Mcl1 (#4572), CHOP (#2895), ATF4 (#11815), pAKT (#4060, Serine‐473 phosphorylation), AKT (#4691), p‐mTOR (#2971), mTOR (# 2972), pERK1/2 (#9101), ERK (#4695), Wee1 (#4936), TRAIL (#3219), and β‐actin (#A2228).
6
Protein Expression Analysis in Gemcitabine-Resistant Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentration of total protein extracted from parental and gemcitabine-resistant cells was determined with a BCA Protein Assay Kit (Pierce, USA). Equal amounts of protein were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, Bedford, USA) using a mini trans-blot (Bio-Rad laboratories, Hercules, CA, USA). Rat anti-human CIP2A, p- AKT(Millipore, Bedford, USA), and BCL2 (Abcam, MA, USA) were used to detect the expression of these proteins. β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as an internal control. Electrochemiluminescence was performed according to the manufacturer's instructions and read with a Chemi lmager 5500 imaging system (Alpha Innotech Co, San Leandro, CA, USA).
7
Western Blot Analysis of Prostate Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from whole-cell lysates using Kinexus Lysis Buffer with Lysis Buffer Cocktail (Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada) and protein concentration was determined using BCA assay (Thermo Scientific, Waltham, MA, USA). Subsequently, 30 μg of total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with antibodies against PRMT6, H3R2me2a (1/500 Novus Biologicals, Littleton, CO), H3K4me3, PSA (1/1500 and 1/6000 Abcam, Cambridge, UK), AR, mTOR (1/1000, Cell Signaling Technology, Inc., Danvers, MA), p21, p27 (1/500, BD Biosciences, Franklin Lakes, NJ), AKT (1/500, Santa Cruz Biotechnologies Inc) and pAKT (1/500, Millipore Billerica, MA), as well as histone H3 (dilution: 1:500, Abcam) and beta-actin (dilution: 1:8000, Sigma-Aldrich) as input controls, as appropriate. Blots were developed using Immun-Star™ WesternC™ Kit (BioRad, Hercules, CA). All experiments were performed in triplicate.
8
Protein Expression Analysis in HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured HUVECs were collected in RIPA buffer to isolate total proteins (110 ). Equal amount of proteins from each sample were loaded onto sodium dodecyl sulfate (SDS) polyacrylamide gels, which were then subjected to electrophoresis. Proteins were then transferred onto PVDF membranes (Bio-Rad), and the following antibodies were employed to detect for the proteins of interest [Cell Signaling Technology: ICAM-1 (4915S, dilution 1:1000), VCAM-1 (13662S, dilution 1:1000), eNOS (32027S, dilution 1:1000), phospho (p)-eNOS (Millipore, 07 -428-I, dilution 1:1000), AKT (4691S, dilution 1:1000), p-AKT (4060S, dilution 1:1000), cleaved-CASPASE3 (9664S, dilution 1:1000), p21 (2947S, dilution 1:1000), and GAPDH (5174S, dilution 1:1000)]. Western blot for FABP3 was performed using polyclonal antibody (ThermoFisher, PA5-92386, dilution 1:1000), and wildtype mouse total heart protein was used as a positive control. Western blots were developed using chemiluminescence substrates (Bio-Rad) and the Licor-Odyssey XF Imaging System. Densitometry was performed to measure the band intensities using the Image Studio Lite.
9
Western Blot Analysis of Apoptotic Markers in 293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 293T cells were seeded on cell culture dishes before transfection. Using Lipofectamine 2000 reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA), cells were transfected with 4 µg of each plasmid according to manufacturer instructions. Assays were performed 48 h after transfection. Western blot analysis included lysates of 293T cells. Hypotonic lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, sodium orthovanadate, sodium fluoride, pH 7.4) was added to each sample. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20, pH 8.0) for 1 h. A monoclonal antibody against Bax, Bcl-2, Caspase-3, Akt, p-Akt (Millipore, Billerica, MA, USA) and Fyn (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as primary antibody at a dilution of 1:300, followed by treatment with horseradish peroxidase-conjugated secondary antibodies and ECL Plus Western Blot detection reagents (Thermo Scientific, Waltham, MA, USA).
10
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The protein concentration was determined using a bicinchoninic acid assay, and equal amounts (20–30 µg) of total protein were separated by 6–10% SDS-PAGE and transferred onto PVDF membranes, which were then blocked with 5% skim milk for 2 h at room temperature. The primary antibodies against ZFX (1:1,000; cat. no. ab115998; Abcam), β-Actin (1:1,000; cat. no. ab8224; Abcam), PI3K (1:1,000; cat. no. 05–212; Merck KGaA), AKT (1:1,000; cat. no. 07-383; Merck KGaA) and p-AKT (1:1,000; cat. no. 04-736; Merck KGaA) were used to incubate the membranes overnight at 4°C. Following incubation with secondary goat anti-mouse (1:2,000; cat. no. ab6789; Abcam) or goat anti-rabbit (1:2,000; cat. no. ab6721; Abcam) antibody for 2 h at room temperature, blots were visualized using ECL Detection Reagent (cat. no. P0018; Beyotime Institute of Biotechnology) and analyzed using Image Lab software (Bio-Rad Laboratories, Inc.).
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!