Journal: Cell Death and Differentiation

Article Title: Chaperone-like protein p32 regulates ULK1 stability and autophagy

doi: 10.1038/cdd.2015.34

Figure Lengend Snippet: p32 interacts with ULK1. (a) HEK293T cells were transiently transfected with the indicated expression constructs. The anti-Myc immunoprecipitates were resolved by SDS-PAGE, and the proteins were visualized by silver staining, and indicated bands were analyzed by mass spectrometry. (b) Western blotting analysis of input and anti-Myc IP derived from HEK293T cells that were transiently transfected with WT or mutant ULK1 (K46I) and p32-Myc. (c) Hela cells expressing Myc-ULK1 were grown either in normal or in EBSS medium for 6 h. Cell lysates were immunoprecipitated with anti-Myc antibody followed by immunoblotting with anti-p32 antibody. (d) The interaction of endogenous ULK1 and p32 was detected in Hela cells. Normal rabbit IgG was used as a negative control for the immunoprecipitation procedure. (e) Purified GST-p32 was incubated with cell lysates derived from HEK293T cells transfected with the indicated HA-ULK1 constructs. Proteins retained on Sepharose were then blotted with the indicated antibodies. (f) Extracts from HEK293T cells transfected with HA-ULK1 were incubated with recombinant full-length (FL) GST-p32 or GST-p32 mutants coupled to GSH-Sepharose. Proteins retained on Sepharose were then blotted with the indicated antibodies. (g) Purified recombinant His-ULK1-CT was incubated with GST-p32. Proteins retained on Sepharose were then blotted with the indicated antibodies. (h) HEK293T cells were transfected with vectors encoding HA-ULK1, Flag-Atg13 and p32-Myc, as indicated. Cell lysates were immunoprecipitated with anti-Myc antibody. Western blotting was performed using the indicated antibodies. (i) Purified His-ULK1-CT or His-p32 was incubated with purified GST-Atg13. Proteins retained on Sepharose were then blotted with the indicated antibodies. (j) Hela cells were fractionated and analyzed by immunoblotting using antibodies against the indicated proteins. Cyto, cytosol; Mito, mitochondria

Article Snippet: Rabbit anti-phosphorylated Atg13 (600-401-C49) antibody was purchased from Rockland (Gilbertsville, PA, USA).

Techniques: Transfection, Expressing, Construct, SDS Page, Silver Staining, Mass Spectrometry, Western Blot, Derivative Assay, Mutagenesis, Immunoprecipitation, Negative Control, Purification, Incubation, Recombinant