Journal: Antioxidants
Article Title: Endosomal H 2 O 2 Molecules Act as Signaling Mediators in Akt/PKB Activation
doi: 10.3390/antiox14050594
Figure Lengend Snippet: Movement of the Akt adaptor protein APPL1 from the cytosol to the early endosome by endosomal H 2 O 2 during EGF activation. ( A ) Schematic representation of APPL1 domains showing three parts (gray boxes): BAR (essential for Rab5-GTP binding and dimerization), pleckstrin homology (PH, participating in association with Rab5-GTP), and phosphotyrosine binding (PTB, required for binding to activated receptors and Akt). ( B ) Cos7 cells were deprived of serum for 5 h and incubated in a high-glucose medium in the presence of GOx (20 mU/mL) for 10 min. Alternatively, the cells were exposed to GOx for 10 min, washed, and incubated in the absence of GOx for another 30 min (wash out). The fixed cells were subjected to immunofluorescence analysis by using the antibodies to APPL1. The regions indicated by the arrows are shown at a higher magnification in the second row. ( C ) Quantitative analysis of the relative fluorescence intensity (RFI) from ( B ). Data are presented as means ± SEM ( n = 4 cells for each condition). ** p < 0.01. ( D ) Cos 7 cells were deprived of serum for 5 h, incubated in the absence (Buffer) or presence of DPI (10 μM) for 30 min or catalase (2 mg/mL), and incubated with additional EGF (200 ng/mL) at the selected time points. Selected snapshot confocal microscopy images of the fixed cells were shown for endogenous APPL1. Arrowheads indicate the areas shown at a higher magnification. ( E ) Quantitative analysis of the RFI from ( D ). Data are presented as means ± SEM ( n = 7 cells for each condition). * p < 0.05, ** p < 0.01. ( F ) Cat-Endo- or catalase-expressing Cos7 cells were deprived of serum for 5 h and stimulated with EGF (200 ng/mL) for 1 min. The fixed cells were subjected to immunofluorescence analysis by using the antibodies to APPL1. The RFI of endosomal APPL1 is presented as means ± SEM ( n = 3 images for each condition). ** p < 0.01. ( G ) HeLa cells were transfected with control siRNA (siCont) or siRNA for APPL1 (siAPPL1) for 72 h and deprived of serum for 5 h. After EGF (200 ng/mL) was added to the cells and left for 1 min, cell lysates were analyzed using antibodies against the proteins listed. ( H ) Quantitative analysis of the relative band intensity of pS 473 Akt and pT 308 Akt from ( G ). Data are presented as means ± SEM ( n = 3 blots). * p < 0.05.
Article Snippet: The pEGFPC1-human APPL1 plasmid (plasmid #22198) was obtained from Addgene (Watertown, MA, USA), and the pHyPer-Cyto (HyPer-C) plasmid was purchased from Evrogen Joint Stock Company (Moscow, Russia).
Techniques: Activation Assay, Binding Assay, Incubation, Immunofluorescence, Fluorescence, Confocal Microscopy, Expressing, Transfection, Control
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