Journal: The Journal of Biological Chemistry
Article Title: MAPK15 is part of the ULK complex and controls its activity to regulate early phases of the autophagic process
Figure Lengend Snippet: MAPK15 participates in the ULK1 complex. A, HEK293 and NIH3T3 were harvested and lysed in 1% Nonidet P-40 lysis buffer. The proteins (10 mg per sample) were then subjected to immunoprecipitation ( IP ) with isotype control IgG ( IgG ) or MAPK15-specific antibody (custom preparation). Total lysates and immunoprecipitated complexes were subjected to Western blotting ( WB ) to detect endogenous ULK1 and MAPK15 protein levels. Representative images from three different experiments are shown ( n = 3). B , HEK293T cells were transfected with plasmid encoding for ULK1-FLAG in combination with HA-MAPK15 WT , HA-MAPK15 KD , or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n = 3). C , HEK293T cells were transfected with different plasmids encoding for FLAG-tagged ULK1, ATG13, FIP200, or ATG101 proteins in combination with HA-MAPK15 or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n = 3). D , HeLa cells stably expressing EGFP-GABARAP were transfected with HA-MAPK15 and ULK1-FLAG plasmids and subjected to immunofluorescence analysis after 48 h. Representative images are from three different experiments ( n = 3). EGFP-GABARAP is visualized in green , ULK1-FLAG in red , and HA-MAPK15 in blue ( upper panels ). White squares indicate zoom areas. Scale bars , 10 μm. E, colocalization rate was measured by LAS AF (Leica Microsystem) software, analyzing single cells that express both MAPK15 and ULK1 in addition to EGFP-GABARAP. Thresholds were set at 20% for each channel as suggested from the manufacturer's protocol. Reciprocal colocalization rate between ULK1 and GABARAP, ULK1 and MAPK15, and GABARAP and MAPK15 were evaluated. Measures were obtained by analyzing at least 100 cells/sample from three different experiments. F , colocalization spots were analyzed for fluorescent signal intensity for each channel; the yellow line indicates the measured points on the merged image .
Article Snippet: Briefly, washed cellular pellet fractions were resuspended in MAPK lysis buffer made of 20 m m HEPES (PAA, ), pH 7.5, 10 m m EGTA (Sigma, E4378), 40 m m β-glycerophosphate (Sigma, G6501), 1% Nonidet P-40 (Sigma, I3021), 2.5 m m MgCl2 (Sigma, M2670), 2 m m orthovanadate (Sigma, S6508), 2 m m NaF (Carlo Erba, 7681494), 1 m m DTT (IBI, IB21040), and protease inhibitors mixture (Roche Diagnostics, 05056489001).
Techniques: Lysis, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Immunofluorescence, Software