Protein Expression Analysis in Mouse Cardiac Tissues

mRNA was extracted from mouse heart tissues and qPCR was performed as described previously49 (link)54 (link). Protein extraction and subcellular fractions were performed as described previously49 (link)54 (link). Targeted proteins including VCP, eNOS and iNOS were detected by western blotting as previously described15 (link)18 (link). The primary antibodies used for western blotting were anti-VCP (catalog no. 2648S, rabbit, dilution of 1:1000, Cell Signaling Technology), anti-iNOS (catalog no. ab136918, rabbit, dilution of 1:1000, Abcam), and anti-eNOS (catalog no. Sc-654, rabbit, dilution of 1:1000, Santa Cruz Biotechnology). Anti-GAPDH (Catalog no. ab9484, mouse, dilution of 1:10,000, Abcam) and anti- voltage-dependent anion-selective channel protein 1(VDAC) (Catalog no. 4866, rabbit, dilution of 1:5,000, Cell Signaling Technology) were used as loading controls.

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Lizano P., Rashed E., Stoll S., Zhou N., Wen H., Hays T.T., Qin G., Xie L.H., Depre C, & Qiu H. (2017). The valosin-containing protein is a novel mediator of mitochondrial respiration and cell survival in the heart in vivo. Scientific Reports, 7, 46324.

Publication 2017

anti-VCP anti-iNOS anti-eNOS

Antibodies Dilution Enos Gapdh Heart Inos Mouse Mrna Proteins Rabbit Subcellular fractions Tissues Voltage dependent anion selective channel protein 1

Corresponding Organization :

Other organizations : Rutgers, The State University of New Jersey, Loma Linda University, University of Alabama at Birmingham

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA expression levels of target genes (measured by qPCR)
Protein expression levels of target proteins (measured by western blotting)

control variables

Experimental procedures for mRNA extraction and qPCR, as described previously in references 49 and 54
Experimental procedures for protein extraction and subcellular fractionation, as described previously in references 49 and 54
Primary antibodies used for western blotting, including anti-VCP, anti-iNOS, and anti-eNOS
Loading control antibodies used for western blotting, including anti-GAPDH and anti-VDAC

controls

Positive controls: None explicitly mentioned
Negative controls: None explicitly mentioned

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