Western Blot Analysis of ONH Astrocytes

Cultured human ONH astrocytes were lysed with lysis buffer as described previously (Noh et al., 2013 (link)). The primary antibodies included rabbit polyclonal anti-NR1 and NR2A antibody (1:1,000; Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-GLAST (EAAT1, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-DRP1 antibody (1:1,000; BD Transduction Laboratories), rabbit polyclonal anti-phospho-DRP1 at Ser616 antibody (1:1,000; Cell Signaling), and mouse monoclonal anti-actin antibody (1:5,000; Millipore). The scanned film images were analyzed with ImageJ (National Institute of Health, MD) and band densities were normalized to the band densities for actin.

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Ju W.K., Kim K.Y., Noh Y.H., Hoshijima M., Lukas T.J., Ellisman M.H., Weinreb R.N, & Perkins G.A. (2014). Increased Mitochondrial Fission and Volume Density by Blocking Glutamate Excitotoxicity Protect Glaucomatous Optic Nerve Head Astrocytes. Glia, 63(5), 736-753.

Publication 2014

rabbit polyclonal anti-NR1 and NR2A antibody mouse monoclonal anti-DRP1 antibody rabbit polyclonal anti-phospho-DRP1 at Ser616 antibody mouse monoclonal anti-actin antibody

Actin Anti antibody Antibodies Antibody Astrocytes Buffer Human Monoclonal antibody Mouse Nr2a Rabbit

Corresponding Organization : University of California, San Diego

Other organizations : Northwestern University

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Variable analysis

independent variables

Lysis of cultured human ONH astrocytes with lysis buffer

dependent variables

Levels of NR1 and NR2A proteins
Levels of GLAST (EAAT1) protein
Levels of DRP1 protein
Levels of phospho-DRP1 at Ser616

control variables

Band densities of actin were used to normalize the protein levels

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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