Quantification of Signaling Proteins

Protein assays (Pierce, Rockford, IL) and Western blot analysis (Bio-Rad, Hercules, CA) were performed as previously described (Cantara et al., 2012 (link)) with 8 μg of protein loaded/well and protein bands quantified using ImageJ (version 1.46r) with normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Levels of each phosphorylated protein were additionally normalized to total protein levels. Antibodies, their phosphorylated epitope (when relevant), their dilution from stock solution, and their source are as follows: GAPDH (1:10,000; Fitzgerald, Acton, MA), p25/TPPP (clone EPR 3316, 1:1000; Epitomics), phospho–LIMK 1 (Thr-508; 1:500; Abcam), phospho–LIMK 2 (T505; 1:500; Abcam), phospho-cofilin (Ser-3; 77G2, 1:1000; Cell Signaling), cofilin (1:2000; Sigma-Aldrich), phospho-FAK (Tyr-576/577; 1:1000; Cell Signaling), phospho-FAK (1:1000; Tyr-397; Cell Signaling), FAK (1:1000; Cell Signaling), phospho-paxillin (Tyr-118; 1:1000; Cell Signaling), paxillin (1:500; Cell Signaling), phospho–histone H3 (pHH3; Ser-10; 1:1000; Cell Signaling), and histone H3 (1:1000; Cell Signaling).

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Ray S., Fanti J.A., Macedo D.P, & Larsen M. (2014). LIM kinase regulation of cytoskeletal dynamics is required for salivary gland branching morphogenesis. Molecular Biology of the Cell, 25(16), 2393-2407.

Publication 2014

Western blot analysis GAPDH phospho–LIMK 1 phospho-cofilin (Ser-3; 77G2, cofilin phospho-FAK (Tyr-576/577; phospho-FAK phospho-paxillin paxillin phospho–histone H3 (pHH3; Ser-10; histone H3

Antibodies Assays Clone Cofilin Dilution Epitope Glyceraldehyde 3 phosphate dehydrogenase Histone h3 Limk 1 Limk 2 Paxillin Protein Western blot analysis

Corresponding Organization :

Other organizations : Albany State University, University at Albany, State University of New York

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of each phosphorylated protein (phospho-LIMK 1, phospho-LIMK 2, phospho-cofilin, phospho-FAK, phospho-paxillin, phospho–histone H3)

control variables

Protein loaded per well (8 μg)
Normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Normalization of phosphorylated protein levels to total protein levels

controls

Positive control: GAPDH
No negative control explicitly mentioned

Annotations

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