Protein Extraction and Immunoblotting from Liver Tissue

Proteins from freshly frozen liver tissues were extracted, segregated through electrophoresis, and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) [27 (link)]. Membranes were incubated with primary antibodies against CD63 (Proteintech, Rosemont, IL, USA), CD9 (Proteintech), phosphorylated-NF-κB (Cell Signaling Technology, Danvers, MA, USA), NF-κB (Cell Signaling Technology), HIF-1α (iReal Technology, Hsinchu, Taiwan), proapoptotic BAX (Abcam) [27 (link)], antiapoptotic Bcl-2 (Cell Signaling) [27 (link)], proapoptotic cleaved Caspase-3 (Cell Signaling) [27 (link)], or actin (internal standard; Sigma-Aldrich). Bound antibodies were detected through chemiluminescence (ECL Plus kit; Amersham). Band density was measured using densitometry (ImageJ).

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Chang C.Y., Chen K.Y., Shih H.J., Chiang M., Huang I.T., Huang Y.H, & Huang C.J. (2021). Let-7i-5p Mediates the Therapeutic Effects of Exosomes from Human Placenta Choriodecidual Membrane-Derived Mesenchymal Stem Cells on Mitigating Endotoxin-Induced Mortality and Liver Injury in High-Fat Diet-Induced Obese Mice. Pharmaceuticals, 15(1), 36.

Publication 2021

nitrocellulose membranes phosphorylated-NF-κB NF-κB ECL Plus kit

Actin Antibodies Bcl 2 Caspase 3 Chemiluminescence Densitometry Electrophoresis Frozen Liver Membranes Nf κb Nitrocellulose Proteins Tissues

Corresponding Organization : Taipei Medical University Hospital

Other organizations : Changhua Christian Hospital, Redcliffe Hospital, University of Queensland

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Variable analysis

independent variables

Protein extraction from freshly frozen liver tissues
Protein segregation through electrophoresis
Protein transfer onto nitrocellulose membranes

dependent variables

Protein levels of CD63
Protein levels of CD9
Protein levels of phosphorylated-NF-κB
Protein levels of NF-κB
Protein levels of HIF-1α
Protein levels of proapoptotic BAX
Protein levels of antiapoptotic Bcl-2
Protein levels of proapoptotic cleaved Caspase-3
Protein levels of actin (internal standard)

control variables

Freshly frozen liver tissues
Nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA)
Primary antibodies against CD63 (Proteintech, Rosemont, IL, USA), CD9 (Proteintech), phosphorylated-NF-κB (Cell Signaling Technology, Danvers, MA, USA), NF-κB (Cell Signaling Technology), HIF-1α (iReal Technology, Hsinchu, Taiwan), proapoptotic BAX (Abcam), antiapoptotic Bcl-2 (Cell Signaling), proapoptotic cleaved Caspase-3 (Cell Signaling), or actin (Sigma-Aldrich)
Chemiluminescence detection (ECL Plus kit; Amersham)
Densitometry measurement (ImageJ)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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