Journal: eLife

Article Title: Crk proteins transduce FGF signaling to promote lens fiber cell elongation

doi: 10.7554/eLife.32586

Figure Lengend Snippet: ( A ) Genetic ablation of Fgfr1 and Fgfr2 disrupted the proper formation of the lens vesicle with the phosphorylation of Erk and Crk/Crkl proteins being noticeably absent. ( B ) Mouse Embryonic fibroblast (MEF) cells treated with FGF2 displayed an increase in pCrk/Crkl and pErk levels, which were abrogated by the removal of Fgfr1 and Fgfr2 using a Cre-expressing adenovirus. ( C ) Ablation of Crk proteins in MEF cells reduced FGF2-induced Erk phosphorylation. The pErk/Erk ratios were noted below the pERK blot. ( D ) The CrkCKO lens displayed a significant decrease in pERK staining compared to the wild type lens.

Article Snippet: Peptide, recombinant protein , recombinant human FGF2 , ScienCell , 104–02 , .

Techniques: Phospho-proteomics, Expressing, Staining

Journal: eLife

Article Title: Crk proteins transduce FGF signaling to promote lens fiber cell elongation

doi: 10.7554/eLife.32586

Figure Lengend Snippet: ( A ) Overexpression of Fgf3 in Fgf3 OVE391 and Fgf3 OVE393A strains resulted in increased lens fiber cell elongation and overall lens sizes. However, both of these phenotypes were suppressed after being crossed with the CrkCKO mutant, indicating a genetic epistasis interaction between the FGF and Crk signaling pathways. ( B ) Quantification of lens fiber cell length. One-way ANOVA test followed by Tukey's multiple comparisons test, *p<0.01, n = 3. ( C ) Deletion of the Crk genes did not prevent the premature differentiation phenotype observed in the Fgf3 overexpressing lens, as indicated by a reduction of lens progenitor cell markers (Ki67, E-cad and Foxe3) and an increase of differentiation cell markers (p57, Jag1, and C-Maf) within the presumptive lens epithelial layer (arrows). Nonetheless, pERK staining was significantly reduced in the CrkCKO; Fgf3 OVE391 lens. ( D ) The Cre-expressing adenovirus induces efficient genetic recombination in lens explant cultures as indicated by the ROSA mTmG reporter. In Crk flox/flox ;Crkl floxflox explants, the Cre-mediated deletion of Crk proteins prevented Fgf2 from inducing cell shape changes. 10.7554/eLife.32586.010 Figure 4—source data 1. Source data for and .

Article Snippet: Peptide, recombinant protein , recombinant human FGF2 , ScienCell , 104–02 , .

Techniques: Over Expression, Mutagenesis, Protein-Protein interactions, Staining, Expressing

Journal: eLife

Article Title: Crk proteins transduce FGF signaling to promote lens fiber cell elongation

doi: 10.7554/eLife.32586

Figure Lengend Snippet: ( A ) FGF2 was still able to induce an upregulation of pCrk/Crkl and pERK in Fgfr1 Crk/Crk ;Fgfr2 flox/flox MEF cells after treatment with a Cre-expressing adenovirus. ( B ) A homozygous Fgfr1 Crk/Crk mutation did not affect pCrk/Crkl staining (arrows) in the lens even after the genetic removal of other Fgf receptors ( Fgfr2-4 ).

Article Snippet: Peptide, recombinant protein , recombinant human FGF2 , ScienCell , 104–02 , .

Techniques: Expressing, Mutagenesis, Staining

Journal: eLife

Article Title: Crk proteins transduce FGF signaling to promote lens fiber cell elongation

doi: 10.7554/eLife.32586

Figure Lengend Snippet: ( A–B ) Mutating the Frs2-bindng site in Fgfr1 ΔFrs resulted in the loss of pCrk/Crkl in the Pax6 Le -Cre;Fgfr1 flox/ΔFrs ;Fgfr2 floxflox mutant lens. ( B ) FGF2 was unable to induce the phosphorylation of Crk proteins in Fgfr1 flox/ΔFrs ;Fgfr2 floxflox MEF cells after treatment with the Cre expressing adenovirus. ( C ) FGF2-induced pCrk/Crkl and pErk were significantly downregulated in both Frs2 and Shp2 null MEF cells. ( D ) Shp2 was successfully depleted in the Frs2CKO;Shp2CKO lens, which resulted in the loss of pCrk/Crkl staining. ( E ) pERK was downregulated in both CrkCKO and Shp2CKO lenses and was further reduced in CrkCKO;Shp2CKO mutants. ( F ) FGF2-induced pCrk/Crkl and pErk were down regulated in Grb2 deficient MEF cells. ( G ) TAP-taged Crkl pulled down Frs2, Shp2 and Grb2 after FGF2 stimulation. Note that only the slower moving phosphorylated form of Frs2 successfully interacted with Crkl.

Article Snippet: Peptide, recombinant protein , recombinant human FGF2 , ScienCell , 104–02 , .

Techniques: Mutagenesis, Phospho-proteomics, Expressing, Staining

Journal: eLife

Article Title: Crk proteins transduce FGF signaling to promote lens fiber cell elongation

doi: 10.7554/eLife.32586

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , recombinant human FGF2 , ScienCell , 104–02 , .

Techniques: Transfection, Construct, Synthesized, Transduction, Recombinant, Plasmid Preparation, In Situ