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AMPK-Mediated Phosphorylation of ATGL-1 Generates More Phospho-(Ser) 14-3-3 Binding motifs and Enhances ATGL-1 Interaction with the C. elegans 14-3-3 Protein, PAR-5.

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posted on 22.06.2015, 04:12 authored by Meng Xie, Richard Roy

(A) An increase in both the abundance and the number of bands that were detected by the P-14-3-3 antibody was observed in lysates obtained from N2 animals treated with the potent phosphatase inhibitor calyculin A. (B) More phospho-(Ser) 14-3-3 binding motif was generated in the ATGL-1 immunoprecipitates from control daf-2 dauers compared to daf-2; aak(0) mutant dauers. Immunoprecipitation of ATGL-1 from total lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were immunoblotted with antisera that recognizes phospho-(Ser) 14-3-3 binding motif. More lysate was loaded for control daf-2 animals to equalize the amount of ATGL-1 protein loaded with that of daf-2; aak(0) animals. “IP:ATGL-1” refers to the protein lysate that was subjected to immunoprecipitation with our anti-ATGL-1 polyclonal antibody. “Input” refers to the total protein lysate before performing the immunoprecipitation step. (C) The anti-PAR-5 antibody recognized a single band at approximately 30kD, corresponding to its predicted molecular weight, which disappeared following par-5(RNAi). (D) PAR-5 protein levels are the same in control daf-2 and daf-2; aak(0) dauers. (E) and (F) Immunoprecipitation of PAR-5 (D) or ATGL-1 (E) from protein lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were subjected to immunoblot analysis using ATGL-1 or PAR-5 antibody as indicated on the panels. More lysate was loaded for control daf-2 animals to equalize the amount of ATGL-1 protein loaded with that of daf-2; aak(0) animals. “IP:ATGL-1” refers to the protein lysate that was subjected to immunoprecipitation with our anti-ATGL-1 polyclonal antibody. “Input” refers to the total protein lysate before performing the immunoprecipitation step.

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