Cellular proteins were isolated from heart tissues (20 (link)), separated by SDS-polyacrylamide gel electrophoresis and transferred onto Hybond enhanced chemiluminescence (ECL) membranes (Amersham Pharmacia, Piscataway, NJ). The ECL membranes were incubated with appropriate primary antibody [anti-phospho-Akt and anti-Akt (Cell Signaling Technology, Beverly, Ma)], followed by an incubation with peroxidase-conjugated second antibodies (Cell Signaling Technology). The membranes were analyzed by the ECL system (Amersham Pharmacia). The signals were detected with the G: BOX system and quantified using GeneTools software (Syngene, Frederick, MD).
Anti phospho akt and anti akt
Manufactured by Cell Signaling Technology
Anti-phospho-Akt and anti-Akt are primary antibodies used in western blotting and immunohistochemistry to detect phosphorylated and total Akt, respectively. Akt is a key protein kinase that plays a central role in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated second antibodies, by Cell Signaling Technology (1 mentions)
Ecl system, by Cytiva (1 mentions)
Genetools software, by Syngene (1 mentions)
Anti cd36, by R&D Systems (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Ecl plus system, by GE Healthcare (1 mentions)
Anti mmp2, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti phospho ikk, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Anti fibronectin, by BD (1 mentions)
Anti phospho ampk and anti ampk, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti srebp 1c, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti phospho akt and anti akt
1
Western Blot Analysis of Phosphorylated Akt
2
Antibody Immunoblotting Assay
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Anti-CD36 (R&D System), anti-phospho AKT and anti-AKT (Cell Signaling Technology), anti-Heat shock protein 70 (HSC70) (Santa Cruz Biotechnology) primary antibodies and the secondary antibody peroxidase conjugate (Santa Cruz Biotechnology) were obtained from commercial sources.
3
Western Blot Quantification of Signaling Proteins
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Western blot analyses of cell lysates were performed as previously described [16 (link)] except that blots were probed with 1:1000-diluted primary antibodies for rabbit anti-MMP-2, MMP-9, anti-phospho-AKT and anti-AKT, anti-phospho-IKK, anti-IκBα, and anti- NF-κB p65 (Cell Signaling, Beverly, MA) or mouse anti-human L1CAM (NeoMarker, Fremont, CA), anti-E-cadherin (Cell Signaling), anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-fibronectin (BD Biosciences, San Jose, CA). For a loading control, blots were probed with an anti-EF1-α monoclonal antibody (1:10,000; R&D Systems, Minneapolis, MN). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, GE Healthcare Life Sciences), signals were detected by an enhanced chemiluminescence (ECL) Plus system (GE Healthcare, Pittsburgh, PA). Protein bands were quantified using ImageJ software.
4
Transcriptional Regulation of Lipid Metabolism
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The RT-qPCR analyses of SREBP-1c, FAS, ACC, PPARα, ACOX and CPT1 were performed as described previously (Park et al. 2016) (link). The quantity of each target mRNA was quantified relative to 36B4 mRNA. The ChIP assays were performed using HepG2 cells as described previously using the anti-SREBP-1c (Santa Cruz) or anti-PPARα antibodies (Abcam). Sequences of primer pairs used for real-time PCR are listed in Table 2 (Park et al. 2016) (link). The immunoblot analyses were performed as described previously using the anti-SREBP-1c (Santa Cruz), anti-phospho-PPARα and anti-PPARα (Abcam), anti-phospho-AMPK and anti-AMPK (Cell Signaling), anti-phospho-ACC and anti-ACC (Cell Signaling), anti-phospho-Akt and anti-Akt (Cell Signaling) and anti-β-actin antibodies (Santa Cruz). The level of each protein was quantified using the ImageJ program and normalized to the level of β-actin.
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