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. 2011 Mar 15;6(3):e16828. doi: 10.1371/journal.pone.0016828

Figure 2. BAG3 recognizes folding status of αB-crystallin.

Figure 2

(A) BAG3 binds to mutant type of αB-crystallin strongly. Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before immunoprecipitation was also loaded to confirm protein expression (right four lanes). (B) Direct recognition of mutant αB-crystallin by BAG3. Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. Mutated αB-crystallin preferentially binds to BAG3. Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).