2D-DIGE analysis of proteomic changes in whole lenses of 14-day-old mice induced by knock-in of the αA-R49C mutation.

<p>(A) A 2D gel of lens proteins labeled with cyanine dyes derived from WT1 proteins labeled with Cy2, WT2 proteins labeled with Cy3, and αA-R49C homozygous lens proteins labeled with Cy5. (B, C) Protein spots that were selected for analysis from the gel in (A) are shown. Proteins were identified by tandem mass spectrometry and Mascot searches of spots that were selected from the gels. Quantitative image analysis and mass spectrometry data for the identified proteins from these gels are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone-0095507-t003" target="_blank">Table 3</a>.</p

Multi-gel analysis of proteins that showed differences in abundance between 2-day-old WT and heterozygous or homozygous αA-R49C lenses.

<p>WT, wild-type.</p

2D-DIGE analysis of proteomic changes in whole lenses of 2-day-old mice with knock-in of the αA-R49C mutation.

<p>(A) 2D gel of cyanine dye-labeled lens proteins derived from wild-type sample 1 (WT1) proteins labeled with Cy2, WT2 proteins labeled with Cy3, and αA-R49C heterozygous proteins labeled with Cy5. (B) 2D gel of cyanine dye-labeled lens proteins derived from WT1 proteins labeled with Cy2, WT2 proteins labeled with Cy3, and αA-R49C homozygous proteins labeled with Cy5. Protein spots that were selected for analysis from the gels shown in (A) and (B) are shown in Fig. S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone.0095507.s005" target="_blank">File S1</a> and were identified by tandem mass spectrometry and Mascot searches. Quantitative image analysis and mass spectrometry data for the identified proteins are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone-0095507-t001" target="_blank">Table 1</a>. Arrows indicate the shift in position of the αA-crystallin bands <i>(red)</i> to a more acidic pI with the mutation.</p

Quantitative analysis of abundance changes in proteins from postnatal 2-day-old WT and αA-R49C knock-in lenses by mass spectrometry.

<p>The 3D data sets for representative proteins in two WT (WT1 and WT2) and one αA-R49C heterozygous or αA-R49C homozygous mutant sample are shown. WT1 and WT2 proteins were labeled with Cy2 and Cy3 dyes, respectively and αA-R49C mutant proteins with Cy5. Fold changes between each sample are indicated on the right. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone-0095507-t001" target="_blank">Table 1</a> for the identity of proteins present in each protein spot.</p

Ingenuity Pathways analysis of lens proteins identified in αA-R49C knock-in mutant lenses.

<p>Analysis of altered protein networks by Ingenuity Pathway software. Biological networks and pathways generated from input data (Wild-type vs. αA-R49C, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone-0095507-t001" target="_blank">Tables 1</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone-0095507-t003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone.0095507.s001" target="_blank">Table S1</a>) indicate proteins with altered abundance in gray. (A) A network with HSPA8 at the hub. (B) A second network highlights Histone H4 at the hub of the protein connectivity map. Additional networks are shown in Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone.0095507.s005" target="_blank">File S1</a>.</p

2D-DIGE analysis of proteomic changes in whole lenses of WT, αA-R49C heterozygous, and αA-R49C homozygous mutant lenses using a pool-based analysis.

<p>(A) WT samples were labeled with Cy2, a pool of all samples (containing WT, αA-R49C heterozygous and homozygous proteins) was labeled with Cy3, and the αA-R49C heterozygous mutant sample was labeled with Cy5. The pool sample was a common comparator for each sample. (B, C) Spots that were selected based on analysis of the gels are shown. Quantitative image analysis by biological variation analysis was performed across several samples, and mass spectrometry data for the identified proteins from these gels are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone-0095507-t003" target="_blank">Table 3</a>.</p

Quantitative analysis of the changes in abundance of proteins in postnatal 14-day-old lens from WT and αB-R120G knock-in mice by mass spectrometry.

<p>The 3D data sets for representative proteins in two WT (WT1 and WT2) and one αB-R120G mutant sample are shown. (A) WT1 and WT2 proteins were labeled with Cy3 and Cy5 dyes, respectively, and αB-R120G heterozygous mutant lenses with Cy2. (B) WT1 and WT2 proteins were labeled with Cy2 and Cy3 dyes, respectively, and αB-R120G homozygous mutant lenses with Cy5. Fold changes between each sample are indicated on the right. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095507#pone-0095507-t004" target="_blank">Table 4</a> for the identity of proteins present in each protein spot.</p

Quantitative analysis of protein abundance in 14-day-old WT, αB-R120G lenses heterozygous and homozygous.

<p>WT, wild-type.</p

L⁵⁰⁰ and L⁵⁰³ are critical for the targeting of Glut4 to GSVs in the basal state.

<p>After co-expression of wild-type Glut4/eGFP and the various mutants within the IRM region of Glut4/Dsred by recombinant adenovirus infection, adipocytes were serum starved for 2 hours, washed with cold PBS, fixed with 4% paraformaldehyde, and then subjected to confocal microscopy analysis. The red color on the left panels represents mutants of Glut4/Dsred, the green color in the middle panels represents wild type Glut4/eGFP, and the yellow color in the right panels represents the colocalization of Glut4/eGFP and mutated Glut4/Dsred. The images were taken approximately through the middle of the cells and are representative of 4–5 independent experiments. The scale bar represents 10 μm.</p

L⁵⁰⁰ and L⁵⁰³ are critical for insulin-stimulated translocation of Glut4 to the cell periphery.

<p>After co-expression of wild-type Glut4/eGFP and the various mutants within the IRM region of Glut4/Dsred using recombinant adenovirus infection, adipocytes were serum starved for 2 hours, stimulated with 1 μM insulin for 30 minutes, washed with cold PBS, fixed with 4% paraformaldehyde, and then subjected to confocal microscopy analysis. The red color in the left panels represents mutants of Glut4-Dsred, the green color in the middle panels represents wild type Glut4/eGFP, and the yellow color in the right panels represents the colocalization of Glut4/eGFP and mutated Glut4/Dsred. The images were taken approximately through the middle of the cells and are representative of 4–5 independent experiments. The scale bar represents 10 μm.</p