Human Kelly neuroblastoma cell lines were obtained from the University of California at San Francisco. Cells were grown in RPMI medium with 10% FBS at 37°C. In most experiments, cells were conditioned in 2% FBS for 5 h and replaced with full medium and recombinant human insulin-like growth factor-I (20 ng/ml) (Invitrogen, Waltham, MA, USA) for 1 h before harvesting. Lysates were collected and sonicated. Primary antibodies were as follows: anti-MYCN, anti-phosphorylated Akt, anti-p-rpS6, anti-Akt, anti-rpS6 (Cell Signaling Technology, Danvers, MA, USA), and β-tubulin (Upstate, Buffalo, NY, USA). Immunoblots were developed with horseradish peroxidase-conjugated secondary antibodies (Calbiochem, San Diego, CA, USA) and Enhanced Chemiluminescence Plus reagents (Amersham, Piscataway, NJ, USA).
Anti phosphorylated akt
Manufactured by Cell Signaling Technology
Sourced in United States, China
Anti-phosphorylated Akt is a laboratory reagent used to detect and quantify the phosphorylation of the Akt protein. Akt is a key signaling molecule involved in various cellular processes, and its phosphorylation is an important indicator of cellular activity and signaling pathways. This reagent can be used in techniques such as Western blotting, immunohistochemistry, and ELISA to measure the levels of phosphorylated Akt in biological samples.
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Lab products found in correlation
Anti akt, by Cell Signaling Technology (30 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (9 mentions)
Anti total akt, by Cell Signaling Technology (8 mentions)
Anti bcl 2, by Cell Signaling Technology (6 mentions)
Anti β actin, by Cell Signaling Technology (6 mentions)
Anti phosphorylated erk1 2, by Cell Signaling Technology (5 mentions)
Anti erk1 2, by Cell Signaling Technology (5 mentions)
Anti egfr, by Cell Signaling Technology (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (3 mentions)
Anti foxo1, by Cell Signaling Technology (3 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (3 mentions)
Anti phosphorylated ampk, by Cell Signaling Technology (3 mentions)
Anti phosphorylated egfr, by Cell Signaling Technology (3 mentions)
Anti bax, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti erk, by Cell Signaling Technology (2 mentions)
Simvastatin, by Merck Group (2 mentions)
57 protocols using anti phosphorylated akt
1
Insulin-like Growth Factor Signaling in Neuroblastoma
2
Flavin Mononucleotide Therapeutic Protocol
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FMN (purity >98.0%) was purchased from MedChem Express, LLC, (New Jersey, USA). MCT, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA) and anti-α-smooth muscle actin (α-SMA) antibody were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). FMN was dissolved in DMSO and diluted with olive oil (45 mg/ml). MCT was dissolved in 1 M HCL neutralized with 1 M NaOH, and diluted with normal saline. Then, the pH was adjusted to 7.2–7.4. Anti-cleaved caspase-3, anti-GAPDH, anti-phosphorylated-AKT, anti-AKT, anti-P-ERK, anti-ERK, anti-rabbit IgG horseradish peroxidase (HRP)-conjugated and anti-mouse IgG HRP-conjugated antibodies were obtained from Cell Signaling Technology, Inc., Danvers, MA, USA. Anti-Bax, anti-Bcl-2, and anti-proliferating cell nuclear antigen (PCNA) antibodies were purchased from Abcam, Cambridge, UK. An H&E assay kit was obtained from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. BCA Protein and Colorimetric TUNEL Apoptosis assay kits were purchased from Beyotime Institute of Biotechnology.
3
Immunoblotting for Synaptic Protein Analysis
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Samples were lysed in TEN buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1% NP-40, supplemented with protease inhibitors and phosphatase inhibitors. Protein concentration was determined by BCA assay (BCA Protein Assay Kit, Thermo Fisher Scientific). Equal amounts of protein samples were subjected to SDS-polyacrylamide gel electrophoresis and PVDF membrane transfer. Proteins were identified by incubating with indicated primary antibodies and then with appropriate HRP-conjugated secondary antibodies. Protein band intensities were determined using the Image J software [45 (link)]. Antibodies used were: anti-GAPDH (Abways, ab0037), anti-PSD93 (Abcam, ab151721), anti-PSD95 (Cell Signaling Technology, 3450 S), anti-Akt (Cell Signaling Technology, 9272 S), anti-phosphorylated Akt (Cell Signaling Technology, 9271 S), and HRP-conjugated secondary antibodies (Thermo Fisher Scientific, 31430 and 31460).
4
Immunoblotting Analysis of EGFR, ERK, and AKT
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A549 and PC-9 cells were lysed in buffer containing proteinase inhibitors. The total protein of cells was obtained using the Nuclear Extract Kit (Active Motif Corp, USA). Protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL, USA). Samples containing 100 µg of total protein were electrophoresed on 10% SDS-PAGE and transferred onto a nitrocellulose membrane by electroblotting. The blots were probed using the following antibodies: anti-EGFR (1∶1000; Santa Cruz, CA), anti-phosphorylated-EGFR (1∶1000), anti-ERK1/2 (1∶600), anti-phosphorylated-ERK1/2 (1∶600), anti-AKT (1∶1000), anti-phosphorylated-AKT (1∶1000), anti-IGF-1R (1∶600), anti-phosphorilated-IGF-1R (1∶600) and anti-β-actin antibody (1∶800) (all the above antibodies were from Cell Signaling Technology, USA), followed by incubation with horseradish peroxidase (HRP) conjugated goat-anti-mouse secondary antibody (Santa Cruz, CA, USA). The blots were visualized by an enhanced chemiluminescence kit (ECL) (Amersham Pharmacia Biotech, Arlington Heights, IL, USA) according to the manufacturer’s instructions. Each experiment was performed in triplicate.
5
Analyzing RNA and Protein Expression Changes
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Total RNA was extracted using ISOGEN (Wako, Osaka, Japan), and real-time RT-PCR was performed as previously described [26] (link). Primer sequences are shown in Table S1 . We normalized the values to that of Gapdh. Western blot analysis was performed using the following antibodies: anti-Akt, anti-phosphorylated Akt, anti-FoxO1, anti-FoxO3a, anti-phosphorylated FoxO1 (Thr24)/FoxO3a (Thr32), anti-JNK, anti-phosphorylated JNK, anti-Mst1, and anti-phosphorylated Mst1 antibodies (Cell Signaling, Danvers, MA); anti-phosphorylated FoxO3a (S207) antibody (Invitrogen, Tokyo, Japan); and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
6
Estrogen-Positive Cell Line Maintenance
7
Measuring Signaling Pathways in Lung Tissue
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The protein extracts of lung were incubated with the primary antibody [anti-phosphoinositide 3-kinases (PI3K) (1:1000; Cell Signaling Technology); anti-nuclear factor kappa B (NFκB, p65) (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.); anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) (1:10000; Abcam plc); anti-phosphorylated IκBα (1:1000; Cell Signaling Technology); anti-VEGF (1:1000; Santa Cruz Biotechnology); anti-VEGFR-1, -phosphorylated VEGFR-1 (1:1000; Abcam plc); anti-VEGFR-2 (1:500; Millipore Corporation); anti-phosphorylated VEGFR-2 (1:1000; Cell Signaling Technology); anti-Rho-associated kinase (RhoA) (1:1000; Cell Signaling Technology); anti-Akt (1:500, Cell Signaling Technology); anti-phosphorylated Akt (1:2000, Cell Signaling Technology); anti-extracellular signal-regulated kinase (ERK), -phosphorylated ERK (1:3000, Millipore Corporation)]. Then the blots were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG antibody, Sigma Chemical Co., St. Louis, MO, U.S.A.). With a computer assisted video densitometer and digitalized software (Kodak Digital ScienceTM ID Image Analysis Software, Eastman Kodak Co., Rochester, NY, U.S.A.), the blots were scanned, photographed then the signal intensity (integral volume) of the appropriate bands were analyzed.
8
Agmatine Modulation of Inflammation
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Agmatine, LPS (Escherichia coli 0111:B4) and protoporphyrinIX zinc(II) (ZnPP)were purchased from Sigma–Aldrich (Saint Louis, MO, USA). LY294002, protease/phosphatase inhibitor cocktail, anti-β-actin, anti-phosphorylated Akt and anti-total Akt antibodies were products of Cell Signaling Technology (Danvers, MA, USA). Idazoxan, anti-Nrf2 and anti-iNOS antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).Anti-HO-1 antibody was product of Abcam (Cambridge, MA, USA). RPMI 1640 and fetal bovine serum were products of Gibco-BRL Invitrogen (San Diego, CA, USA). Yohimbine, efaroxan, dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich. BCA protein assay kit, total nitric oxide assay kit and nuclear and cytoplasmic protein extraction kits were from Beyotime (Jiangsu, China).
9
Molecular Mechanisms of MALP-2 Signaling
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MALP-2 was purchased from Enzo Lifesciences (ALX-162-027-C050; Plymouth Meeting, PA). Anti-HO-1, anti-phosphorylated and anti-total c-Src, anti- phosphorylated Btk, anti-phosphorylated Akt and anti-total Akt antibodies were products of Cell Signaling Technology (Beverly, MA). Anti-total Btk and anti-Mal were from Abcam (Cambridge, MA). Anti-MyD88, anti-p85α and anti-Nrf2 antibodies were purchased from Santa Cruz (Santa Cruz, CA). Cy3-conjugated goat anti-rabbit IgG antibody was purchased from Invitrogen (Frederick, MD). Anti-TLR2 and anti-TLR6 neutralizing antibodies, dominant negative (DN) plasmids DN-TLR2 and DN-TLR6, and DN-Mal and DN-MyD88 were products of InvivoGen (San Diego, CA). Protein phosphatase 1 (PP1) was from Enzo Lifesciences (Plymouth Meeting, PA). LFM-A13 and LY294002 were obtained from Calbiochem (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO), respectively. Complete protease inhibitor cocktail was obtained from Roche Applied Science (Mannheim, Germany).
10
Immunoblotting Analysis of Signaling Pathways
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Stimulation, extraction, SDS–PAGE, and immunoblotting were performed as previously described (Martin et al, 2014 ; Izawa et al, 2017 ). The following antibodies were used for immunoblotting: antiphosphorylated tyrosine (4G10; dilution 1:1,000), antiphosphorylated PLC‐γ1 (#2821S; dilution 1:1,000), anti‐PLC‐γ1 (#2822S; dilution 1:1,000), antiphosphorylated ERK 1/2 (#4376S; dilution 1:1,000), anti‐ERK 1/2 (#4695S; dilution 1:1,000), antiphosphorylated P38 (#4511S; dilution 1:1,000), antiphosphorylated AKT (serine 473, 4058S; dilution 1:1,000), and anti‐Ku70 (4103S; dilution 1:1,000) purchased from Cell Signaling Technology; anti‐CTPS1 (EPR8086B; dilution 1:1,000) purchased from Abcam; and anti‐actin (A2066; dilution 1:1,000) purchased from Thermo Fischer Scientific; anti‐RASGRP1 antibodies from Merck Millipore (MABS146, RASGRP1 epitope recognized unknown; dilution 1:1,000), from Thermo Fischer Scientific (PA5‐25750, raised against 495–521 amino acids of RASGRP1; dilution 1:1,000), and from Abcam (EPR9609, raised against the N‐terminal part of RASGRP1; dilution 1:1,000). Membranes were then washed and incubated with anti‐mouse or anti‐rabbit HRP‐conjugated antibodies from Cell Signaling (dilution 1:10,000). Pierce ECL Western blotting substrate was used for detection.
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