Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Detection of K2-acetylated γD-crystallin in the human lens. Western blot analysis of γD-crystallin and N ε -acetyllysine-modified proteins in the human lens. Water-soluble human lens proteins were subjected to Western blot analysis using a monoclonal antibody against γD-crystallin (A). The membrane was stripped and reprobed using a monoclonal antibody against N ε -acetyllysine (B). Densitometry of Western blot B is shown in C. Water-soluble human lens proteins were immunoprecipitated using a monoclonal antibody against γD-crystallin and were subjected to Western blot analysis using an antibody against N ε -acetyllysine (D). The age of the donor lenses is shown below the lanes. M denotes the molecular weight markers. Arrows indicate the positions of the light (LC) and heavy chains (HC) of the antibody. The (−) denotes nonacetylated recombinant γD-crystallin; the (+) denotes in vitro acetylated recombinant γD-crystallin. SDS-PAGE of the purified γD-crystallin is shown in panel E. Lanes 1 and 2 are two preparations of γD-crystallin, and lane 3 is in vitro acetylated γD-crystallin. Western blot analysis of acetylated γD-crystallin using an antibody against N ε -acetyllysine; acetylation was carried out using various molar excess concentrations of Ac 2 O relative to lysine in γD-crystallin (F).

Article Snippet: The membrane was reprobed using a monoclonal antibody against N ε -acetyllysine (1:2500 dilution, Cell Signaling Technologies, Danvers, MA).

Techniques: Western Blot, Modification, Immunoprecipitation, Molecular Weight, Recombinant, In Vitro, SDS Page, Purification