Monoclonal mouse anti‐PTEN antibody (clone 6H2.1) was from Cascade Bioscience (Winchester, UK). Purified rabbit anti‐phospho‐PTEN (Ser380/Thr382/Thr383) antibody, rabbit anti‐pan‐Akt antibody, rabbit anti‐phospho‐Akt (Thr308) antibody, rabbit anti‐phospho‐Akt (Ser473) antibody, rabbit anti‐FAK antibody, and rabbit anti‐phospho‐FAK (Tyr397) antibody were from Cell Signaling Technology (Boston, MA, USA). Purified anti‐fibronectin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Purified and FITC mouse anti‐E‐cadherin antibody and anti‐β‐catenin antibody were from BD Biosciences (San Diego, CA, USA). Streptavidin (SAv)‐Alexa 594 (SAv‐594)‐conjugated anti‐mouse antibody was from Invitrogen Life Technologies (Carlsbad, CA, USA). Affinity‐isolated rabbit anti‐actin antibody was from Sigma‐Aldrich (St. Louis, MO, USA). Can Get Signal was from Toyobo (Tokyo, Japan). Doxycycline, pTet‐On Advanced, pTRE‐Tight Vector, and Adeno‐X Expression System 3 were from Clontech Laboratories (Mountain View, CA, USA). PhosSTOP was from Roche Applied Science (Mannheim, Germany). Hoechst33342 was from Dojindo (Kumamoto, Japan). 1,2,4,5‐Benzenetetramine tetrahydrochloride (FAK inhibitor 14) was from Tocris Bioscience (Bristol, UK).
Rabbit anti phospho akt ser473 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-phospho-Akt (Ser473) antibody is a primary antibody that specifically recognizes the phosphorylated form of the serine 473 residue in the Akt protein. It is used to detect and quantify the activation of the Akt signaling pathway.
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Lab products found in correlation
Anti β catenin antibody, by BD (1 mentions)
Doxycycline, by Takara Bio (1 mentions)
Phosstop, by Roche (1 mentions)
Ptre tight vector, by Takara Bio (1 mentions)
Ptet on advanced, by Takara Bio (1 mentions)
Rabbit anti fak antibody, by Cell Signaling Technology (1 mentions)
Adeno x expression system 3, by Takara Bio (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
Can get signal, by Toyobo (1 mentions)
Mouse anti α tubulin antibody, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Rabbit anti total akt antibody, by Cell Signaling Technology (1 mentions)
Tissuelyser 24 grinder, by Jingxin (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Mouse anti akt antibody, by Cell Signaling Technology (1 mentions)
Mtt reagent, by Merck Group (1 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Oxyresveratrol oxy, by Merck Group (1 mentions)
Rabbit anti phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
7 protocols using rabbit anti phospho akt ser473 antibody
1
Antibody-Based Protein Analysis Protocol
2
Quantifying Akt Phosphorylation in Larval Tissues
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Larval fat body tissues were homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCl, pH 7.4) using a Tissuelyser-24 grinder (Jingxin, Shanghai, China). After centrifugation at 15,000 g at 4 °C for 20 min, the supernatants were subjected to separation by SDS-PAGE before immunoblotting analysis. The primary antibodies used are rabbit anti-phospho-Akt (Ser473) antibody (1:1000, Cell Signaling, Catalogue no.9271s), rabbit anti-total-Akt antibody (1:1000, Cell Signaling, Catalogue no.9272), mouse anti-mCherry (1:3000, Abbkine, Catalogue no. A02080), mouse anti-α-tubulin antibody (1:4000; Sigma, Catalogue no. T6199). Anti-secondary antibodies are anti-rabbit IgG (1:2000, Cell Signaling, Catalogue no. 5151) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:2000, Cell Signaling, Catalogue no. 7076). Non-saturated bands were quantified on FIJI-ImageJ (National Institutes of Health) and presented as a ratio in relation to total-Akt. At least three biological replicates were quantified.
3
Oxyresveratrol Regulates Cell Cycle and Apoptosis in HaCaT Keratinocytes
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Human immortalized keratinocytes (HaCaT) were purchased from Cell Lines Service GmbH (Eppelheim, Germany). Recombinant human TNF-α was purchased from PeproTech (Rocky Hill, NJ, USA). Oxyresveratrol (OXY) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Guava® Cell Cycle Reagent was purchased from Luminex Corporate (Austin, TX, USA). Rabbit anti-phospho-AKT (Ser473) antibody, mouse anti-AKT antibody, rabbit anti-phospho-GSK3-β (Ser9), mouse anti-GSK3-β antibody, rabbit anti-Ki-67 antibody, rabbit anti-MCL-1 antibody, DAPI (4′, 6-diamidino-2-phenylindole, dihydrochloride), and LY294002 (a specific inhibitor of the PI3K/AKT) were purchased from Cell Signaling Technology (Boston, MA, USA). Goat anti-mouse IgG-IRDye®800CW and goat anti-rabbit IgG-IRDye®680RT were purchased from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit conjugated with Alexa488 was purchased from Thermo Fisher Scientific (Waltham (HQ), MA, USA).
4
Western Blot Analysis of Akt and S6 Phosphorylation
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Western blot analysis was performed as previously described [25 (link)]. Briefly, after lysis and determination of protein concentration, samples were separated on a 12% SDS-polyacrylamide electrophoresis gel and transferred onto a Hybond ECL nitrocellulose membrane (Amersham Biosciences, Freiburg, Germany). Proteins were visualized by ECL western blotting detection reagents (Amersham Biosciences), according to manufacturer's instruction, and following antibodies were used: rabbit anti–phospho-Akt (Ser473) antibody (Cell Signaling), rabbit anti-phosph-Akt (Thr308) antibody (Cell Signaling), mouse anti-Akt antibody (Bioscience, Heidelberg, Germany), rabbit anti–phospho-S6 ribosomal protein (Ser235/236) antibody (Cell Signaling), rabbit anti–S6 ribosomal protein antibody (Cell Signaling), or mouse anti-GAPDH antibody (Cell Signaling) followed by goat-anti-mouse IgG or goat-anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Heidelberg, Germany).
5
Liver Western Blot Analysis
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Two livers from each group were combined into one sample and total protein was extracted for western blot analysis (one sample mixed with liver tissues from two fish, n = 8).
Western blotting was performed as described previously [83 (link)]. The antibodies used were as follows: rabbit anti-β-actin antibody (1:2000, Proteintech, Wuhan, China), rabbit anti-α-SMA antibody (1:1000, Proteintech, Wuhan, China), rabbit anti-SIRT1 (1:1500, Proteintech, Wuhan, China), rabbit anti-NOX4 antibody (1:1000, Proteintech, Wuhan, China), rabbit anti-NRF2 antibody (1:1500, Proteintech, Wuhan, China), rabbit anti-phospho-AKT (Ser473) antibody (1:2000, Cell Signaling), rabbit anti-phospho-AMPK (Thr172) antibody (1:1000, Cell Signaling), rabbit anti-AMPK antibody (1:1000, Proteintech, Wuhan, China). Protein expression was normalized to that of β-actin.
Western blotting was performed as described previously [83 (link)]. The antibodies used were as follows: rabbit anti-β-actin antibody (1:2000, Proteintech, Wuhan, China), rabbit anti-α-SMA antibody (1:1000, Proteintech, Wuhan, China), rabbit anti-SIRT1 (1:1500, Proteintech, Wuhan, China), rabbit anti-NOX4 antibody (1:1000, Proteintech, Wuhan, China), rabbit anti-NRF2 antibody (1:1500, Proteintech, Wuhan, China), rabbit anti-phospho-AKT (Ser473) antibody (1:2000, Cell Signaling), rabbit anti-phospho-AMPK (Thr172) antibody (1:1000, Cell Signaling), rabbit anti-AMPK antibody (1:1000, Proteintech, Wuhan, China). Protein expression was normalized to that of β-actin.
6
Immunoblotting Analysis of ErbB2 Signaling
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Mouse anti-ErbB2 (A-2), anti-ErbB2 (9G6), anti-Vinculin antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Rabbit anti-PARP antibody was purchased from Proteintech (Wuhan, China). Rabbit anti-phospho-Akt (Ser473) antibody was purchased from Cell Signaling Technology. Secondary goat anti-mouse and anti-rabbit, donkey anti-goat antibodies were obtained from LICOR. Neratinib (HKI-272) and lapatinib (GW572016) were purchased from Selleck. Oleic acid (OA) and lovastatin were obtained from MeilunBio (Dalian, China). Filipin was obtained from Sigma.
7
Quantitative Immunoblotting of Inflammatory Biomarkers
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Cell supernatants and membranes (25 μg protein) were resolved by SDS-PAGE and electrotransferred to nitrocellulose membranes. Detection was performed with: monoclonal antibodies against nCRP (clone 1D6) and mCRP (clone 8C10), rabbit TF antibody (American Diagnostica), rabbit anti-phospho-Akt Ser473 antibody, rabbit anti-Akt antibody (Cell Signaling), rabbit anti-phospho-Ets-1 Thr38 antibody (Novus Biological), and mouse Ets-1 (1G11) (Abcam). Band densities were determined with the ChemiDoc™ XRS system (Bio-Rad) in chemiluminescence detection mode and analyzed with Quantity-One software (Bio-Rad).
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