Protein Expression Analysis in Kidney

Kidney lysates were prepared and Western blots performed as previously described^{19 (link)}. Quantification of Western Blots was by densitometry with analysis using Image J software^{20 (link)}. The following antibodies were used in the Western Blot analysis: anti-α-Smooth Muscle Actin—FITC antibody (Sigma-Aldrich, St. Louis, USA), anti-Fluorescein-POD Fab fragments (from goat Roche Applied Science, Indianapolis, USA), anti-GAPDH antibody (Rabbit monoclonal antibody, Cell Signaling technology, Massachusetts, USA), anti-Fibronectin antibody (Rabbit monoclonal antibody, Sigma-Aldrich, St.Louis, USA), anti-phospho-PFKFB2 (Ser⁴⁸⁵) (Rabbit monoclonal antibody, Cell Signaling Technology, Massachusetts, USA), anti-PFKFB2 antibody (Rabbit polyclonal antibody, abcam, Cambridge, UK), anti-PKM2 antibody (Rabbit monoclonal, Cell Signaling technology, Massachusetts, USA), anti-pPKM2 antibody (Rabbit polyclonal, Cell Signaling technology, Massachusetts, USA) and anti-rabbit Immunoglobulin HRP-linked (Swine polyclonal, Dako, Agilent Pathology Solutions, Santa Clara, USA).

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Lee M., Harley G., Katerelos M., Gleich K., Sullivan M.A., Laskowski A., Coughlan M., Fraser S.A., Mount P.F, & Power D.A. (2020). Mutation of regulatory phosphorylation sites in PFKFB2 worsens renal fibrosis. Scientific Reports, 10, 14531.

Publication 2020

anti-α-Smooth Muscle Actin—FITC antibody anti-GAPDH antibody anti-Fibronectin antibody anti-PKM2 antibody

Actin Anti immunoglobulin Antibodies Antibody Densitometry Fab fragments Fibronectin Fitc Fluorescein Gapdh Goat Kidney Monoclonal antibody Rabbit Smooth muscle Swine Western blot analysis

Corresponding Organization :

Other organizations : Austin Health, University of Queensland, Mater Research, Translational Research Institute, Monash University, University of Melbourne

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Variable analysis

independent variables

Kidney lysates preparation and Western blots as previously described

dependent variables

Quantification of Western Blots by densitometry with analysis using Image J software
Protein expression levels of α-Smooth Muscle Actin, GAPDH, Fibronectin, phospho-PFKFB2 (Ser485), PFKFB2, PKM2, and pPKM2

control variables

Antibodies used in the Western Blot analysis: anti-α-Smooth Muscle Actin—FITC antibody, anti-Fluorescein-POD Fab fragments, anti-GAPDH antibody, anti-Fibronectin antibody, anti-phospho-PFKFB2 (Ser485) antibody, anti-PFKFB2 antibody, anti-PKM2 antibody, anti-pPKM2 antibody, and anti-rabbit Immunoglobulin HRP-linked

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