Total protein was extracted from CCA cell lines using 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH7.4 containing phosphatase inhibitor, protease inhibitor, and 5 μM PUGNAc. Protein (30 μg) was solubilized in the sample loading medium (SM; 62.5 mM Tris-HCl pH 6.8, 2% SDS, 5% β-ME, 10% Glycerol, and 0.01% Bromphenol blue), separated by SDS-PAGE according to Laemmli38 (link), and electro-transferred onto a PVDF membrane using Bolt and Marhoney transferring buffer (40 mM Tris base, 20 mM sodium acetate, 2 mM EDTA, pH 7.4, 20% methanol, and 0.05% SDS)39 (link). Immunodetection of particular proteins was performed using specific monoclonal antibodies as follow; 1:200 of anti-OGT and anti-MMP7 (Santa Cruz Biotechnology, Santa Cruz, CA); 1:400 of anti-β-catenin (BD Transduction Laboratories, New Jersey); 1:1000 of anti-NF-κB (p65) (Santa Cruz), anti-β-tubulin (Santa Cruz, CA), anti-O-GlcNAc (RL2, Pierce Biotechnology, IL), anti-Akt and anti-pAkt (Cell Signaling Technology, Danvers, MA). The immunoreactivity was detected with the ECLTM Prime Western Blotting Detection System (AmershamTM, Buckinghamshire, UK) and analyzed using an ImageQuant LAS 4000 mini image analyzer and ImageQuant™ TL analysis software (GE healthcare, Buckinghamshire, UK).
Anti akt and anti pakt
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Akt and anti-pAkt are antibodies used for the detection and analysis of Akt and phosphorylated Akt (pAkt) in biological samples. Akt is a serine/threonine protein kinase that plays a crucial role in various cellular processes, such as cell proliferation, survival, and metabolism. The anti-Akt antibody recognizes the total Akt protein, while the anti-pAkt antibody specifically detects the phosphorylated form of Akt. These antibodies are valuable tools for researchers studying the Akt signaling pathway and its role in cellular function and disease.
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Lab products found in correlation
Anti egfr, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti o glcnac, by Thermo Fisher Scientific (1 mentions)
Imagequant tl analysis software, by GE Healthcare (1 mentions)
Anti β catenin, by BD (1 mentions)
Anti β tubulin, by Santa Cruz Biotechnology (1 mentions)
Imagequant las4000 mini image analyzer, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Ecl prime reagent, by GE Healthcare (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti igf 1r, by Cell Signaling Technology (1 mentions)
Anti erk and anti perk, by Cell Signaling Technology (1 mentions)
Anti ampk and anti pampk, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Anti lc3b 1 2, by Cell Signaling Technology (1 mentions)
9 protocols using anti akt and anti pakt
1
Protein Expression Analysis in CCA Cells
2
Comprehensive Protein Extraction and Analysis
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Cells were lysed using RIPA buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 0.25% deoxycholate) supplemented with protease inhibitor tablets (Roche Molecular Biochemicals) and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4 and 1 mM β-glycerophosphate). Cell lysates were cleared by centrifugation (13,000 × g, 15 min, 4 °C), and protein concentration was determined by the Bradford method (Bio-Rad protein Assay). Proteins samples were separated on SDS-PAGE, and membranes were incubated with primary antibodies overnight at 4 °C on a shaker with the following primary antibodies: anti-IGF-1R (Cell Signaling), anti-IR-α (Cell Signaling), anti-EGFR (Cell Signaling), anti-furin (Proteintech), anti-Akt and anti-pAkt (Cell Signaling), anti-ERK and anti-pERK (Cell Signaling), anti-S6 and anti-pS6 (Cell Signaling), anti-S6K1 and anti-pS6K1 (Cell Signaling), anti-AMPK and anti-pAMPK (Cell Signaling) and anti-Tubulin (Sigma). Membranes were washed with TBS-Tween, and incubated with anti-rabbit or anti-mouse secondary antibodies. Detection was carried out using ECL Prime reagent (GE Healthcare).
3
Protein Extraction and Western Blot Analysis
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Total protein was isolated from the primary cardiomyocytes using RIPA buffer (Beyotime Biotechnology, Shanghai, China) with the protease inhibitor cocktail. Protein concentration was quantified with BCA protein assays kit (Beyotime Biotechnology, Shanghai, China). 30-70 μg of proteins was separated by SDS-PAGE gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked by 5% nonfat dry milk with PBS containing 0.1% Tween-20 at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase- (HRP-) conjugated secondary antibodies at room temperature for 1 h. The primary antibodies used in this study were listed as follows: anti-Rap1GAP (1 : 10000, Abcam, Cambridge, UK), anti-LC3B I/II (1 : 1000; Cell Signaling Technology, MA, USA), anti-P62 (1 : 1000; Cell Signaling Technology), anti-Akt and anti-p-Akt (1 : 1000; Cell Signaling Technology), anti-mTOR and anti-p-mTOR (1 : 1000; Cell Signaling Technology), anti-P70s6k and anti-p-P70s6K (1 : 1000; Cell Signaling Technology), and GAPDH (1 : 1000; Cell Signaling Technology). Protein levels were normalized to GAPDH, and the results of signals were quantified by the software of ImageJ.
4
Western Blot Analysis of Protein Expression
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Proteins from whole cell extracts were detected by Western blot according to standard protocols. Primary antibodies: anti-EGFR, anti-pEGFR, anti-ERK1/2, anti-pERK, anti-AKT and anti-pAKT (Cell Signaling Technology), anti-p53 (Novocastra), anti-p21 (Pharmingen), anti-actin (Sigma-Aldrich). Secondary antibodies: anti-mouse and anti-rabbit antibodies (LI-COR Biosciences). The Odyssey® CLx Infrared Imaging System (LI-COR Biosciences) was used for signal detection. Relative signal intensities were given as the quotient of [phospho-protein / unphosphorylated proteins]. Cetuximab-treated samples were normalized to untreated ones and erlotinib-treated samples to DMSO-treated ones.
5
Investigating HPV-Positive Cervical Cancer Cells
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HeLa (HPV18-positive), CaSki (HPV16-positive), Siha (HPV16-positive) human cervical cancer cells and 293 T human embryonic kidney cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). HeLa, CaSki, and Siha cells were cultured in α-MEM supplemented with 10% foetal bovine serum (FBS) and antibiotics, at 37 °C in a humidified 5% CO2 incubator, and 293 T cells were maintained in DMEM culture medium. Western blotting and immunocytofluorescence analysis were conducted using the following antibodies: anti-Axl, anti-MZF1, anti-Sp1, and anti-LaminlB1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HPV16E6 (Full Moon Biosystems, Sunnyvale, CA, USA); anti-AKT and anti-p-AKT (Cell Signaling Technology, Beverly, MA, USA); anti-PTEN and anti-p-PTEN (R&D Systems, Abingdon, UK); anti-MAGI-2 (Novus Biologicals, Littleton, CO, USA); and α-tubulin (Sigma, St. Louis, MO, USA). Horse radish peroxidase (HRP)-conjugated anti-goat IgG and anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies. Soluble Fas ligand (sofas L) was purchased from Enzo life science (CA, USA).
6
Protein Extraction and Western Blot Analysis
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Protein was collected from the supernatant of breast cancer cells lysed on ice in RIPA lysis buffer (Beyotime, Shanghai, Chain) containing 1% protease and 0.1% phosphatase inhibitors. Western blot was performed as described previously [14 (link)]. Specific primary antibodies include anti-hTIPE3 (1:1000, Sigma, USA) and anti-flag (1:1000, Sigma, USA), anti-pAKT and anti-AKT, anti-IkBα and anti-pIkBα, anti-p-P65 and anti-P65 antibodies (1:1000, Cell Signaling Technology, Beverly, USA), anti-β-actin (1:1000, ZSGB-BIO), anti-β-tubulin antibody and anti-LaminA antibody (Sino Biological Inc, China).
7
Protein Extraction and Western Blot Analysis
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Protein extracts from cells were obtained as previously described [61 (link)]. Briefly, proteins samples were extracted by homogenization using a TissueLyser II (QIAGEN, Tokio, Japan) in cold RIPA buffer (200 mM Tris/HCl (pH 7.4), 130 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) SDS, 1% (v/v) Triton X-100, 10 mM MgCl2) with anti-proteases and anti-phosphatases (Sigma-Aldrich; St Louis, MO, USA). Twenty-five μg cell lysates and the VEs obtained from the same amount of cells per plate from at least three independent experiments were separated in 10% SDS-PAGE gels and electroblotted onto nitrocellulose membranes as previously described [13 (link)]. Primary anti-PAkt, and anti-Akt were purchased from Cell Signaling Technology (MA, USA); anti- Alix, anti-Ceruloplasmin, anti-TSG101, anti-CD81, anti-Syntenin, and anti-Mimecan from Sta. Cruz Biotechnology (CA, USA); anti-TGFBeta Ig-h3 (NMP2-67186; dilution 1:500) from Novus Biologicals (NovusBio, CO, USA) and anti-Perilipin-1 from Abcam. Data were expressed as percentages corrected towards GADPH (arbitrary units) in Western blots with mean ± SEM. Data analyses were conducted using GraphPad Prism 6 software.
8
Protein Expression Analysis of Neuronal Cells
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After the treatment, primary cortical neurons were washed and lysed with RIPA lysis buffer (Beyotime, China). The solution was centrifuged at 14000 x g at 4°C for 10min. Concentration of the protein was measured by the BCA protein assay (Beyotime, China). Extracts were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, USA). Membranes were probed with primary antibodies at 4°C overnight (anti-pPI3K and anti-PI3K, 1:1000, Abcam; anti-pAKT and anti-AKT, 1:1000, Cell Signaling Technology; anti-GAP-43, 1:1000, Cell Signaling Technology; anti-GAPDH, 1:500, Boster; anti-activated-caspase-3, 1:500, Beyotime). Band intensities were analyzed with the image J software (1.46r).
9
Comprehensive Protein Analysis Workflow
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Western blot analysis was performed as described previously (Dellambra et al., 2000) , using the following antibodies: anti-CSA H-266, anti-p16INK4a (N20), anti-p63 (4A4), anti-IkBa (C21), antilamin B (M20), and anti-GAPDH (FL-335), all from Santa Cruz Biotechnology (Santa Cruz, CA); anti-pAkt and anti-Akt from Cell Signaling (Leiden, The Netherlands).
For NF-kB activation quantification, nuclear extracts were submitted to the ELISA-based "NFKB Transcription Factor Assay Kit" to detect the activated NF-kB subunit bound to a specific oligonucleotide (TransAm, Carlsbad, CA) .
For cytokine secretion analysis, keratinocytes were cultured for 72 hours in starvation medium. Media were collected and submitted to three biotin-label-based antibody arrays for profiling of protein secretion (RayBio C-Series Human Cytokine Antibody Array C6, C7, and MMP, RayBiothec, Norcross, GA). Protein levels were evaluated by densitometric analysis using a GS-710 scanner and Quantity One software (Bio-Rad, Hercules, CA). Data analysis was performed as described previously (Huang et al., 2010) .
Quantitative RT-PCR RNA was extracted from cells using the TRIzol (Invitrogen). For mRNA quantification, total RNA was reverse transcribed using an oligo(dT) primer (Invitrogen). mRNA levels were analyzed as described in Maurelli et al. (2016) . Primers are shown in Supplementary Table S2 online.
For NF-kB activation quantification, nuclear extracts were submitted to the ELISA-based "NFKB Transcription Factor Assay Kit" to detect the activated NF-kB subunit bound to a specific oligonucleotide (TransAm, Carlsbad, CA) .
For cytokine secretion analysis, keratinocytes were cultured for 72 hours in starvation medium. Media were collected and submitted to three biotin-label-based antibody arrays for profiling of protein secretion (RayBio C-Series Human Cytokine Antibody Array C6, C7, and MMP, RayBiothec, Norcross, GA). Protein levels were evaluated by densitometric analysis using a GS-710 scanner and Quantity One software (Bio-Rad, Hercules, CA). Data analysis was performed as described previously (Huang et al., 2010) .
Quantitative RT-PCR RNA was extracted from cells using the TRIzol (Invitrogen). For mRNA quantification, total RNA was reverse transcribed using an oligo(dT) primer (Invitrogen). mRNA levels were analyzed as described in Maurelli et al. (2016) . Primers are shown in Supplementary Table S2 online.
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