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Akt 4685

Manufactured by Cell Signaling Technology
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AKT; 4685S is a lab equipment product provided by Cell Signaling Technology. It is an antibody that can be used to detect the AKT protein.

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Lab products found in correlation

G box, by Syngene (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) P akt 4060 t, by Cell Signaling Technology (1 mentions) Ecl substrate, by Solarbio (1 mentions) Phospho akt ser473 4060, by Cell Signaling Technology (1 mentions) Gapdh 5174, by Cell Signaling Technology (1 mentions) β actin 4970, by Cell Signaling Technology (1 mentions) Pparγ 2435, by Cell Signaling Technology (1 mentions) Protease inhibitors cocktail, by Roche (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Phosstop, by Roche (1 mentions) Ripa buffer, by Boston BioProducts (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions) Ciap1, by Cell Signaling Technology (1 mentions) Ciap2, by Cell Signaling Technology (1 mentions) Tri methyl histone h3 lys27, by Cell Signaling Technology (1 mentions) Phospho stat3, by Cell Signaling Technology (1 mentions) Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions)

Spelling variants of this product

Anti-Akt (4685) (5 citations)

8 protocols using akt 4685

1

NMU, AKT, and ERK Protein Analysis

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Total protein was extracted from harvested cells using radioimmunoprecipitation assay lysis buffer (R0020; Solarbio; Shanghai, China). Protein samples were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, and then incubated with a specific antibody at 4°C overnight: NMU (24862-1-AP; Proteintech; Rosemont, IL, USA), protein kinase B (AKT; 4685S; Cell Signaling Technology [CST]; Danvers, MA, USA), phosphorylated AKT (p-AKT; 4060S; CST), extracellular signal-regulated kinase (ERK; 4695S; CST), and phosphorylated ERK (p-ERK; 4370S; CST). Then, the PVDF membranes were incubated with secondary antibodies, including goat anti-rabbit immunoglobulin G (IgG[H+L])-HRP (UT2001; UTIBODY; Tianjin, China) at room temperature for 1 h. An antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control (R5174; CST).
Zeng Y., Ma W., Li L., Zhuang G., Luo G., Zhou H., Hao W., Liu Y., Guo F., Tian M., Ruan X., Gao M, & Zheng X. (2023). Identification and validation of eight estrogen-related genes for predicting prognosis of papillary thyroid cancer. Aging (Albany NY), 15(5), 1668-1684.
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2

Quantification of AKT Phosphorylation in Hippocampal Cells

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Mixed primary hippocampal cells were harvested in RIPA buffer containing phosphatase and protease inhibitors. Cells were further lysed using polytron agitation. Protein levels were quantified using a BCA assay and a microplate reader. Western blots were used to quantify differences in protein expression. Samples were run in duplicate within and across gels and were averaged. Proteins were assessed with the following antibodies: AKT #4685S 1:1000 and pAKT #4051S 1:1000 (Cell Signaling Technology, Inc., Danvers, MA). Blots were developed with chemiluminescence and digitally imaged on a scanner (G-Box; Syngene, Frederick, MD). Gray values were obtained using the ImageJ gel analysis tool (Version 1.46r; Wayne Rasband, National Institute of Health, Rockville, MD). For each blot, mean gray value of pAKT and AKT were normalized to a saline-treated sample, and pAKT was divided by AKT to generate ratios.
Frazier H.N., Anderson K.L., Maimaiti S., Ghoweri A.O., Kraner S.D., Popa G.J., Hampton K.K., Mendenhall M.D., Norris C.M., Craven R.J, & Thibault O. (2018). Expression of a Constitutively Active Human Insulin Receptor in Hippocampal Neurons Does Not Alter VGCC Currents. Neurochemical research, 44(1), 269-280.
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3

Western Blot Analysis of Cell Signaling

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Primary antibodies against AKT (#4685S), p-AKT (#4060T), β-catenin (#8480T), CD63 (#55051S), CD81 (#56039) and CD9 (#13403) were purchased from Cell Signaling Technology, USA. The HRP-labeled anti-rabbit IgG monoclonal antibody (#D110065, Sangon Biotech, China) was used as the secondary antibody, and the fluorescent signal was developed with ECL substrate (#SW2010, Solarbio, China). The chemiluminescent signals were captured using a Bio-Rad ChemiDoc XRS system (Bio-Rad, USA).
Jin Y., Meng Q., Zhang B., Xie C., Chen X., Tian B., Wang J., Shih T.C., Zhang Y., Cao J., Yang Y., Chen S., Guan X., Chen X, & Hong A. (2021). Cancer-associated fibroblasts-derived exosomal miR-3656 promotes the development and progression of esophageal squamous cell carcinoma via the ACAP2/PI3K-AKT signaling pathway. International Journal of Biological Sciences, 17(14), 3689-3701.
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4

Antibody Immunoblotting for Insulin Signaling

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An antibody against IRS1 (sc‐8038, 1 : 1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho‐IRS1 (Ser612; 3193S, 1 : 1000), phospho‐AKT (Ser473; 4060S, 1 : 1000), AKT (4685S, 1 : 1000), β‐actin (4970S, 1 : 1000), GAPDH (5174S, 1 : 1000), and PPAR‐γ (2435S, 1 : 1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). GPR50 (19762‐1‐AP, 1 : 1000) antibody was purchased from Proteintech (Rosemont, IL, USA).
Yao Z., Meng J., Long J., Li L., Qiu W., Li C., Zhang J.V, & Ren P. (2022). Orphan receptor GPR50 attenuates inflammation and insulin signaling in 3T3‐L1 preadipocytes. FEBS Open Bio, 13(1), 89-101.
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5

Western Blot Analysis of Cell Signaling

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Cells were lysed in RIPA buffer (Boston Bio Products) supplemented with 1x protease inhibitors cocktail and 1x PhosSTOP (Roche). Thirty microgram of protein were resolved on 10% NuPAGE Bis-Tris gels (Life Technologies) and transferred to nitrocellulose membranes (Bio-Rad). The following primary antibodies were used in this study: cIAP1 (7065), cIAP2 (3130), Phospho-STAT3 (Tyr705; 9145), STAT3 (4904), Ezh2 (5246), Tri-Methyl-Histone H3 (Lys27; 9733), AKT (4685), Phospho-AKT (Ser473; 9271), and β-Actin (3700) (Cell Signaling Technology); Anti-KMT6/EZH2 (phospho S21; ab84989) (Abcam); sheep anti-mouse IgG-HRP, donkey antirabbit IgG-HRP (Amersham Pharmacia Biotech).
da Hora C.C., Pinkham K., Carvalho L., Zinter M., Tabet E., Nakano I., Tannous B.A, & Badr C.E. (2019). Sustained NF-κB-STAT3 signaling promotes resistance to Smac mimetics in Glioma stem-like cells but creates a vulnerability to EZH2 inhibition. Cell Death Discovery, 5, 72.
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6

Western Blot Analysis of Cell Signaling Proteins

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Cells were homogenized on ice in radioimmunoprecipitation assay (RIPA) buffer supplemented with Halt Protease and Phosphatase Inhibitor cocktail (ThermoFisher Scientific) and centrifuged to remove debris. Protein concentration was determined using the Pierce BCA protein Kit (ThermoFisher Scientific). Western blot analysis was conducted using 50 μg of proteins on ExpressPlus precast gels (Genescript). Proteins were blotted onto polyvinylidene difluoride membranes (Millipore). The membranes were probed with the indicated antibodies (RB #9313, pRB #3590, cyclin D1 #2978, ciclin D3 #2936, cyclin E1 #20808, pAKT #4060, AKT #4685, Actin #3700, Tubulin #2146, all from Cell Signaling). All the primary antibodies were used at a dilution of 1:1000 in 5% BSA in TBS-T. Signals were detected with HRP-conjugated secondary antibodies (ThermoFisher Scientific) and the chemiluminescence substrate Luminata Crescendo (EMD Millipore).
Wong K., Di Cristofano F., Ranieri M., De Martino D, & Di Cristofano A. (2019). PI3K/mTOR inhibition potentiates and extends palbociclib activity in anaplastic thyroid cancer. Endocrine-related cancer, 26(4), 425-436.
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7

Detailed MPTP Neurotoxicity Protocol

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MPTP was purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-TH (F-11, sc-25269) and dopamine transporter (DAT, sc-32258) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-BDNF (ab108319), SHDA (ab137040), and SDHB (ab178423) antibodies were purchased from Abcam (Cambridge, MA, USA); anti-Bax (#14796), Phospho-PI3 Kinase (#17366), PI3 K (#4249), Phospho-Akt (#4060), and Akt (#4685) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); Nrf2 (16396-1-AP), Bcl-2 (16396-1-AP), and GAPDH (60004–1) antibodies were purchased from the Proteintech Group (Rosemont, IL, USA); DyLight 488 goat anti-mouse IgG (H + L) (70-GAM4882) and DyLight 594 goat anti-rabbit IgG (H + L) (70-GAR5942) were purchased from Multi Sciences (Hangzhou, China); horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were purchased from Beyotime Biotechnology (Shanghai, China); and the GSH and GSSG Assay Kit (#S0053) and Cu/Zn-SOD and Mn-SOD Assay Kit with WST-8 (#S0103) were purchased from Beyotime Biotechnology (Shanghai, China).
Zhang W., Chen S., Huang X., Tong H., Niu H, & Lu L. (2023). Neuroprotective effect of a medium-chain triglyceride ketogenic diet on MPTP-induced Parkinson’s disease mice: a combination of transcriptomics and metabolomics in the substantia nigra and fecal microbiome. Cell Death Discovery, 9, 251.
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8

Evaluation of EGCG, Quercetin, and Docetaxel Effects on Prostate Cancer Cells

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LAPC-4-AI and PC-3 cells were treated with vehicle control, 40μM EGCG+5μM Q, 5nM Doc, or EGCG+Q+Doc for 48h. Total protein was extracted using RIPA buffer (Santa Cruz Technology, CA). The procedure for Western blot analysis was described before [31 ]. Briefly, 50 μg of protein was separated on a 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA). Proteins were electrotransferred to nitrocellulose membranes. Membranes were incubated with primary anti-human antibodies for the detection of Bax (sc-493), Bcl-2 (sc-509), MRP1 (sc-7773, Santa Cruz Technology), Akt (4685), p-Akt (Ser473, 4058), STAT3 (9132), and p-STAT3 (4058, Cell Signaling Technology, Danvers, MA). GAPDH protein was used as loading control. Protein was visualized and analyzed using a ChemiDoc XRS chemiluminescence detection and imaging system (Bio-Rad Laboratories, Irvine, CA).
Wang P., Henning S.M., Heber D, & Vadgama J.V. (2015). Sensitization to docetaxel in prostate cancer cells by green tea and quercetin. The Journal of nutritional biochemistry, 26(4), 408-415.
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