Anti pakt473

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-pAKT473 is a primary antibody that recognizes the phosphorylated form of the AKT kinase at serine 473. This antibody is commonly used in western blotting and other immunodetection applications to analyze the activation status of the AKT signaling pathway.

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PAKT473 (31 citations)

7 protocols using anti pakt473

Molecular Mechanisms of Senescence in Neurons

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PFT-α was obtained from Sigma-Aldrich China (Shanghai, China; P4236). The TBK inhibitor BX-795 and the Akt inhibitor MK-2206 were purchased from Selleck chemicals (Houston, TX, USA) (s1274 and s1078). Senescence β-gal staining kit (Cell Signaling Technology, Beverly, MA, USA; #9860).
The following antibodies were used in western blot: anti-TBK1 (Abcam, Cambridge, MA, USA, ab-40676, 1:1000), anti-p16 (Abcam, ab-51243, 1:1000), and anti-cyclin A (Santa Cruz, Dallas, TX, USA; sc-596, 1:1000), anti-pAkt 473 (Cell Signaling Technology; #4060, 1:2000), anti-Bmi (Cell Signaling Technology; #648, 1:1000), anti-phosphorylation serine (Boster, China, BM1622), anti-p53 (Santa Cruz; sc-126, 1:1000), anti-β-actin (Santa Cruz; sc-47778, 1:1000). The following antibodies were used in immunofluorescence: anti-NeuN (Abcam, ab-177487, 1:200), anti-Brn-3a (Santa Cruz; sc-8429, 1:200), anti-p16 (Abcam, ab-51243, 1:100), anti-TBK1 (Abcam, ab-40676, 1:200). anti-pAkt 473 (Cell Signaling Technology; #4060, 1:200) was used in immunohistochemistry.

Li L.U., Zhao Y, & Zhang H. (2017). P16INK4a upregulation mediated by TBK1 induces retinal ganglion cell senescence in ischemic injury. Cell Death & Disease, 8(4), e2752-.

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Inhibition of Akt and Necroptosis Pathways

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The following materials were purchased from commercial companies: TNFα (PeroTech; Rocky Hill, NJ, USA); pan-caspase inhibitor z-VAD-fmk (Abcam, Cambridge, MA, USA). InSolution Akt Inhibitor viii isozyme-selective, Akti-1/2 and InSolution rapamycin were obtained from Calbiochem (San Diego, CA, USA). MitoSox Red was obtained from Invitrogen (Carlsbad, CA, USA). Hoechst 33258, butylated hydroxyanisole (BHA) and rotenone were obtained from Sigma (St. Louis, MO, USA). Nec-1 (5-(7-chloro-1H-indol-3-ylmethyl)-3-methylimidazolidine-2,4-dione), the inactive analog of necrostatin-1 analog (Nec-1i; 5-(7-chloro-1H-indol-3-ylmethyl)imidazolidine-2,4-dione),^{3 (link), 12 (link), 13 (link)} was a kind gift from Dr. Greg Cuny. LOX-1 was obtained from Chembridge (compound 5680672; San Diego, CA, USA), and Bai was from Cayman Chemicals (Ann Arbor, MI, USA). Antibodies were obtained from commercial sources: anti-pAkt-473, anti-p–Akt-308, anti-p-GSK-3β, anti-p-mTOR, anti-p-S6, anti-mTOR, anti-Akt1, anti-Akt2, anti-Akt3, and anti-Akt from Cell Signaling (Danvers, MA, USA); anti-RIPK1 from BD Transduction Laboratories (Lexington, KY, USA); anti-RIPK3 from ProSci Incorporated (San Diego, CA, USA); anti-HMGB1 and β-actin were obtained from Abcam.

Liu Q., Qiu J., Liang M., Golinski J., van Leyen K., Jung J.E., You Z., Lo E.H., Degterev A, & Whalen M.J. (2014). Akt and mTOR mediate programmed necrosis in neurons. Cell Death & Disease, 5(2), e1084-.

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Immunoblotting Assay for Protein Analysis

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Following antibodies were used: anti-FBXO31 (F4431), anti-FLAG (F1840), anti-phosphoserine (P5747), anti-phosphothreonine (P6623) and anti-tubulin (T5168) from Sigma. Anti-FBXL20 (TA306520) and anti-DDK (TA50011) from Origene. Anti-myc (#11667149001) from Roche. Anti-His (sc-8036), anti-cleaved PARP-1(sc-56196), anti-HA (sc-7392), anti-BIM (sc-374358), anti-GFP (sc-9996), anti-p53 (sc-126) anti-GSKa/b (sc-7291) from Santacruz. Anti-PUMA (#4976), BAX (#2772), anti-AKT1 (#9272), anti-pAKT473 (#4058), anti-cleaved Caspase-9 (#9501), anti-K48-ubiquitin (#8081), anti-GST(#2624), anti-BCL2 (#4223) from Cell Signaling technology.

Manne R.K., Agrawal Y., Malonia S.K., Banday S., Edachery S., Patel A., Kumar A., Shetty P, & Santra M.K. (2021). FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX. The Journal of Biological Chemistry, 297(4), 101253.

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Evaluating AKT and MEK Signaling

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Anti-SCOP/PHLPP1 (COSMO Bio; Carlsbad, CA, USA), anti-pAKT473, anti-pAKT308, anti-AKT Total, anti-pMEK, anti-MEK Total, anti-α-Tubulin (Cell Signaling Technology; Danvers, MA, USA).

Jackson T.C., Dixon C.E., Janesko-Feldman K., Vagni V., Kotermanski S.E., Jackson E.K, & Kochanek P.M. (2018). Acute Physiology and Neurologic Outcomes after Brain Injury in SCOP/PHLPP1 KO Mice. Scientific Reports, 8, 7158.

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Molecular Pathway Analysis Protocol

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CPT was purchased from Selleckchem company (Houston, TX, USA). Anti-STAT3, anti-p-STAT3 (705), anti-AKT, anti-p-AKT (473), anti-IKB, and anti-p-IKB (32) were from Cell Signaling Technologies (Danvers, MA, USA). Anti-GAPDH was from KangChen company (Shanghai, China).

Jiaojiao S., Yuping H., Yajuan L., Guangyi L., Qiuhong L., Shengbiao L, & Hong Y. (2021). A System Bioinformatics Approach Predicts the Molecular Mechanism Underlying the Course of Action of Radix Salviae Reverses GBM Effects. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 1218969.

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Antibodies for Apoptosis Pathway Analysis

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Mouse anti-caspase-9, anti-caspase-3, anti-Bax, anti-AKT, anti-pAKT(473) and anti-PARP antibodies were purchased from Cell Signaling Technology. Mouse anti-b-actin, anti-Bcl-xL and anti-FLAG monoclonal antibodies were from Sigma. Anti-Msi1 antibody was from Abcam. The antibodies anti-Bid, anti-PTEN, anti-K-RAS, anti-p38, anti-ERK1 and p-ERK1/2 were obtained from Sangon. Anti-pSTAT3(705) and anti-NOXA antibodies were from SANTA CRUZ. Anti-caspase-8 and anti MCL-1 antibodies were from Boster. The Annexin V-FITC apoptosis detection kit and TRAIL were purchased from Merck Millipore and R&D Systems respectively.

Liu N., Chen T., Wang X., Yang D., Xue B, & Zhu H. (2015). Msi1 confers resistance to TRAIL by activating ERK in liver cancer cells. FEBS letters, 589(8).

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Protein Expression Analysis in VSMCs

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The protein levels of Rac1, MYH, SM22α, Akt, p-Akt (308) and p-Akt (473) in VSMCs were analysed by Western blot. Cells were lysed in RIPA Lysis Buffer (Beyotime Institute of Biotechnology, China), and protein was quantified using BCA protein assay reagent (Beyotime). Proteins (25 µg) were resolved on 6% or 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes, which were blocked with 5% BSA. The primary antibodies anti-Rac1/2/3(1:1000), anti-p-Akt (308; 1:1000), anti-p-Akt (473; 1:1000) (all from Cell Signaling Technology, USA), anti-MYH (1:500), anti-SM22α (1:1000) and anti-TRPC1 (1:1000) (all from Santa Cruz, USA) were incubated with polyvinylidene fluoride membranes overnight at 4°C. After successive washes, membranes were incubated for 1 h with HRP-conjugated sheep anti-rabbit IgG (1:1000) and horse anti-mouse IgG (1:1000) at room temperature (Zhongshan Golden Bridge Biotechnology Company, China). Enhanced chemiluminescence developing methods (Beyotime) were used to visualize immunolabelling. Image-Pro Plus was used to take measurements of band density.

Yin Q., Jiang D., Li L., Yang Y., Wu P., Luo Y., Yang R, & Li D. (2017). LPS Promotes Vascular Smooth Muscle Cells Proliferation Through the TLR4/Rac1/Akt Signalling Pathway. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 44(6).

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