For intracellular cytokine staining, murine cells were restimulated after 72 h of culture with PMA (50 ng ml−1), Ionomycin (1 µg ml−1, both from Sigma-Aldrich) and brefeldin A (5 µg ml−1; Biolegend) for 4 h and fixed with 2% para-formaldehyde. Staining of transcription factors was performed without restimulation using the FOXP3/Fixation-Kit (eBioscience, 00–5521–00). Following antibodies were used for murine cell-analysis: anti-CD8a (eBiosciences, 53–6.7), anti-CD44 (eBiosciences, IM7), anti-CD62L (eBiosciences, MEL-14), anti-IFN-y (Biolegend, XMG1.2), anti-IL-17A (eBiosciences, eBio17B7), anti-RORyt (eBiosciences, B2D) or anti-T-BET (eBiosciences, eBio4B10). The cells were measured on FACSCalibur or FACSAria™ III (both BD) and analyzed with FlowJo Software (FlowJo LLC). For phospho-flow the cells were harvested after 48 h of culture, rested for 4 h and treated with 100U/ml rhIL-2 for 2 h, fixed and permeabilised using Lyse/Fix-Buffer (557870, BD) and Perm-BufferIII (558050, BD) then stained with anti-P-STAT5Y694 (eBiosciences, SRBCZX), anti-P-AKTS473 (Cell Signaling, D9E), anti-P-AKTY308 (Cell Signaling, D25E6), anti-P-FOXO1/3a (Cell Signaling, #9464) or anti-P-S6S235/236 (Cell Signaling, D57.2.2E).
Anti pakt s473
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-pAKT S473 is a laboratory reagent that can be used to detect and quantify the phosphorylation of AKT (protein kinase B) at serine 473. It is designed for use in various research applications, such as Western blotting and immunohistochemistry, to study cellular signaling pathways involving AKT.
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72 protocols using anti pakt s473
1
Intracellular Cytokine and Phospho-flow Analysis
2
Western Blot Analysis of Cellular Proteins
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Lysates were resolved on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with anti-vinculin (Sigma, 1:10000), anti-ATP6V1A (Abnova, 1:1000), anti-ATP6V0a3 (Novus, Centennial, CO; 1:1000), anti-FLAG (Sigma, 1:2000), anti-GFP (Cell Signaling, 1:1000), anti-T7 (Novagen, 1:10000), anti-tubulin (Sigma, 1:10000), anti-Rac1 (BD Transduction Laboratories, 1:1000), anti-AKT (Cell Signaling, 1:1000), anti-pAKT (S473) (Cell Signaling, 1:500), anti-ERK2 (EMD Millipore, 1:2000), anti-pERK1/2 (Cell Signaling, 1:1000), anti-SLC4A7 (Santa Cruz, 1:200), or anti-KRas (Santa Cruz, 1:500) primary antibodies followed by Alexa Fluor 680 goat anti-mouse IgG (Life Technologies, 1:10000) or IRDye 800CW goat anti-rabbit IgG (Li-Cor, 1:10000) secondary antibodies. Blots were analyzed using an Odyssey Classic imager (Li-Cor).
3
Liver Pathology Analysis via Immunohistochemistry
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Mice were sacrificed by carbon dioxide asphyxiation. Livers were fixed in 4% formalin overnight, embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin (H&E) for pathology. Liver sections were de-waxed, rehydrated and stained using standard immunohistochemistry protocols32 (link). The following antibodies were used: anti-Pten (Cell Signaling, 9559, 1:100), anti-pAkt S473 (Cell Signaling, 4060, 1:50), β-Catenin (BD,610154, 1:100), anti-p53 (CM5, 1:300), anti-GS (BD 610517, 1:200) and anti-Ck19 (Abcam, ab133496, 1:100). The number of hepatocytes was quantified from >3 low-magnification fields per mouse with 5 mice per group. Immunofluorescence was performed as previously described32 (link). β-Catenin (BD,610154) and phospho-β-Catenin (Abcam, ab53050) antibodies were used. Slides were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). Images were obtained with a Nikon A1R laser scanning confocal microscope using a 40× APO Fluor objective (NA 0.65).
4
Antibody Validation for HPV16 Proteins
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Goat polyclonal anti-HPV16 E6 (sc-1584) and mouse monoclonal anti-HPV16 E7 (sc-6981) were purchased from Santa Cruz (Dallas, TXs, USA). Mouse monoclonal anti-β-actin (A2228) was purchased from Sigma Aldrich (St. Louis, MO, USA). Rabbit monoclonal anti-LDHA (#3582), anti-HK2 (#2867), anti-Glut1 (#12,939), anti-Akt (#9272), and anti-p-Akt S473 (#4060) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Akt inhibitor, LY294002 (#9901) was purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). 5-FU was purchased from Sigma Aldrich (St. Louis, MO, USA). Transfection reagents were purchased from Invitrogen (Carlsbad, CA, USA).
5
Western Blotting Procedure for Protein Analysis
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Western blot analysis was conducted as previously described (Timper et al., 2017 (link)). In brief, membranes were blocked with 10% WB-blocking reagent (Roche, Switzerland) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), and incubated with primary antibodies, made up in TBS-T (0.1% Tween-20) and 5% Western Blotting-Reagent, at 4°C overnight. The following primary antibodies and dilutions were used: anti-GLP-1R (1:1000, #NBP1-97308, Novus Biologicals), anti-Akt (#4686, Cell Signaling), anti-p-Akt(S473) (#4060, Cell Signaling), anti-NDUFA9 (1:1000, #459100, Invitrogen), anti-NDUFB8 (1:1000, #459210, Invitrogen), anti-Complex II 70 kDa Fp Subunit (1:1000, #459200, Invitrogen), anti-UQCRC1 (1:1000, #459140, Invitrogen), anti-Complex IV (1:1000, #459110, Invitrogen), anti-ATP synthase subunit β (1:1000, #21351, Invitrogen), anti-p-Ser-226-GLUT-1 (#ABN991, Merck Millipore), anti-GLUT-1 (#07-1401, Merck Millipore), anti-FGF21 (#AF3057, R&D Systems) and, as loading control: anti-calnexin (1:4000, #208880, Merck Milipore). Quantification of chemiluminescent signals was performed with the ImageJ (Fiji)-software package (Schneider et al., 2012 (link)).
6
Immunoblotting Protocol for Cell Signaling
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For immunoblotting, RIPA buffer with protease inhibitors was used to extract protein from cells and anti-GUCY1A1 (Cayman), anti-GUCY1B1, anti-ERG (Santa Cruz), anti-AKT, anti-pAKT(S473), anti-VEGF (Cell Signaling), anti-HA (Sigma), anti-β-actin (Abcam), or anti-β-tublin (Upstate) antibodies were used. Gels shown are representative of at least three independent experiments.
7
Insulin-Stimulated AKT and ERK Signaling
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Differentiated cells cultured in DMEM/F12 medium were serum starved overnight and then washed with PBS and stimulated with or without 200 nM insulin for 1 h in DMEM/F12 (without glucose or serum) at 37 °C in 5% CO2. The total protein was harvested in RIPA buffer supplemented with protease and phosphatase inhibitors, as described above. The primary antibodies (all used at 1:1000 dilution unless otherwise stated) used were anti pAKT (S473; Cell Signaling) and anti Akt (9272 - 1:5,000 dilution; Cell Signaling), and the secondary antibody used was goat pAb to Rb igG (Ab205718 - 1:20,000 dilution; Abcam). Protein expression studies were also performed by WES, an automated capillary-based size separation and nano-immunoassay system (ProteinSimple, San Jose CA, USA—a Bio-Techne Brand), according to manufacturer’s protocol, for analysis performed using the Compass for Simple Western software v.4.0. The WES was performed on SGBS samples (1:100) for anti pAKT (S473; Cell Signaling), anti Akt (9272 - 1:100 dilution; Cell Signaling), anti pERK (9102; Cell Signaling) and anti ERK (9101 - 1:100 dilution; Cell Signaling).
8
FAK Copy-Gain Detection in Breast Cancer
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A modified real competitive PCR (mrcPCR) was performed to detect the FAK-copy-gain of breast cancer cell lines as previously described [10 (link)].
Reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (real-time qPCR) were performed according to methods described previously [39 (link)]. GAPDH was used as a control for the level of expression. The primers utilized were as follows: FAK (forward: 5’-TGA GAT CCT GTC TCC AGT CTA CAG-3’, reverse: 5’-CAG TAC CCA TCT ATT AGG TCA GCC-3’), GAPDH (forward: 5’-TGA TGA CAT CAA GAA GGT GGT GAA-3’, reverse: 5’-TCC TTG GAG GCC ATG TGG GCC AT-3’).
Western blotting was performed according to a method described previously [39 (link)]. Anti-FAK (#05-537), anti-pFAK (Y397, #05-1140), and anti-cleaved caspase-3 (#AB3623) were purchased from Millipore (Billerica, MA, USA), and anti-β-actin antibody (#A2228) was acquired from Sigma-Aldrich. Anti-Caspase-3 (#9662), anti-AKT (#9272), and anti-pAKT (S473) antibodies were obtained from Cell Signaling (Danvers, MA, USA).
Reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (real-time qPCR) were performed according to methods described previously [39 (link)]. GAPDH was used as a control for the level of expression. The primers utilized were as follows: FAK (forward: 5’-TGA GAT CCT GTC TCC AGT CTA CAG-3’, reverse: 5’-CAG TAC CCA TCT ATT AGG TCA GCC-3’), GAPDH (forward: 5’-TGA TGA CAT CAA GAA GGT GGT GAA-3’, reverse: 5’-TCC TTG GAG GCC ATG TGG GCC AT-3’).
Western blotting was performed according to a method described previously [39 (link)]. Anti-FAK (#05-537), anti-pFAK (Y397, #05-1140), and anti-cleaved caspase-3 (#AB3623) were purchased from Millipore (Billerica, MA, USA), and anti-β-actin antibody (#A2228) was acquired from Sigma-Aldrich. Anti-Caspase-3 (#9662), anti-AKT (#9272), and anti-pAKT (S473) antibodies were obtained from Cell Signaling (Danvers, MA, USA).
9
Comprehensive Immunoblotting Profiling of Cellular Signaling
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Five-7 x 106 cells were prepared by lysis in a buffer made up of 20 mM Tris, 150 mM NaCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 2 mM EGTA (ethylene glycol-tetra-acetic acid), 0.5% v/v Triton X-100 supplemented with protease inhibitor cocktail (Sigma), 1 mM DTT (dithiothreitol; Amersham Biosciences), 1 mM PMSF (phenyl-methyl-sulfonyl fluoride; Sigma), 1 mM okadaic acid (Sigma) and phosphatase inhibitor cocktail (Thermo Scientific). Proteins were subjected to SDS-PAGE, transferred to PVDF membranes and immunoblotted with the following primary Abs: anti-CK2α (provided by Dr. M. Ruzzene, University of Padova, Italy), anti-CK2β, anti-RELA, anti-FOXO1 (Abcam), anti-pRELA S529 (recognizes S527 in mouse), anti-IRF4, anti-BLIMP-1, anti-BCL6 (Santa Cruz), anti-GAPDH (Ambion), anti-pAKT S129 (provided by Dr. M. Ruzzene, University of Padova, Italy), anti-AID (Invitrogen), anti-NOTCH2 (D76A6), anti-pAKT S473, anti-AKT, anti-pERK1/2 T202/Y204, anti-pBTK Y223, anti-ERK1/2, anti-pPTEN S380/T382/T383, PTEN, anti-pFOXO1 S256, anti-pSTAT6 Y705, anti-STAT6 (Cell Signaling). Secondary Abs: anti-rabbit IgG HRP-linked Ab (Cell Signaling), HRP labeled goat anti-mouse IgG (KPL), goat anti-rat IgG HRP-conjugated (Calbiochem), donkey anti-goat IgG HRP-conjugated (Santa Cruz).
10
Comprehensive Western Blot Analysis
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Western blot assay was conducted as previously described [24 (link)]. Antibodies used in this study were as follows: anti-GAPDH (10494-1-AP, Proteintech), anti-AKT (C67E7, Cell Signaling Technology), anti-pAKT (S473) (193H12, Cell Signaling Technology), anti-PI3K (Y388, Abcam), anti-PI3K (p110) (Y384, Abcam), anti-S1PR3 (EPR454, Abcam), anti-EGR2 (EPR4004, Abcam), anti-IGF2BP1 (22803-1-AP, Proteintech), anti-IGF2BP2 (11601-1-AP, Proteintech), anti-IGF2BP3 (14642-1-AP, Proteintech), anti-WTAP (10200-1-AP, Proteintech).
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