Journal: Redox Biology

Article Title: PER1 interaction with GPX1 regulates metabolic homeostasis under oxidative stress

doi: 10.1016/j.redox.2020.101694

Figure Lengend Snippet: PER1 Binding to GPX1 Mediates Diurnal GPx Activity . A, B) Representative co-immunoprecipitation results of PER1 and GPX1 interaction from cytoplasmic extracts from WT (A) and Per1 −/− (B) mouse liver or small intestine at ZT1 or ZT13. β-actin as an input control. C) Molecular docking of GPX1 to PER1 and the backbone of PER1 and GPX-1 are shown in blue and red (C, top), respectively. Location of key residues was identified by mutagenesis (C, bottom). D-E) Representative immunoprecipitation analysis of PER1 and GPX1 interaction from lysates from NIH-3T3 cells. Lysates from NIH-3T3 cells expressing HA-tagged PER1 or mutant forms of PER1 (178–324 aa deletion and 1056–1177 aa deletion as CK) were immunoprecipitated with an anti-HA antibody (D). Lysates from NIH-3T3 cells coexpressing HA-tagged fragments of GPX1 and a vector expressing PER1 were immunoprecipitated with an anti-HA antibody (E). *GPX1 residues 1–136 are not detected because the UGA stop codon terminates translation in advance while recoded as selenocysteine (46 aa) in full-length conditions. F) In vitro a supplement of the fragment PER1 208–414 increases Gpx activity in Per1 −/− mice (n = 4 per group, *p < 0.05, **p < 0.01 versus β-actin group). β-actin, fragment PER1975-1290 (4 μg/ml) and Proteinase K (PK) digested the fragment PER1 208–414(4 μg/ml) products were performed as negative controls. G, H) Representative immunofluorescence images of GPX1 (Alexa Fluor 568-labeled) and Per1-GFP in NIH-3T3 cells (G). MitoTracker Red CMXRos-labeled mitochondria and Per1-GFP staining in NIH-3T3 cells (H). Images were obtained using a Delta Vision OMX system (GE Healthcare) and processed using GE SoftWoRx Imaging Analysis software. Bars indicate 1 μm in length. Throughout, male mice for all experiments were maintained on standard chow. Data are presented as the mean ± SEM and analyses were performed using one-way ANOVA for F. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Then, samples were imaged using a Delta Vision OMX system (GE Healthcare), and processed using GE SoftWoRx Imaging Analysis software.

Techniques: Binding Assay, Activity Assay, Immunoprecipitation, Mutagenesis, Expressing, Plasmid Preparation, In Vitro, Immunofluorescence, Labeling, Staining, Imaging, Software

Journal:

Article Title: Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric G?s Protein

doi: 10.1091/mbc.E04-06-0446

Figure Lengend Snippet: Interaction between Hrs and Gαs. (A) In vitro-translated, 35S-labeled Gαs binds to GST-Hrs but not to GST alone. GST-Hrs and GST proteins (∼75 μg each) immobilized on glutathione beads were incubated with in vitro-translated, [35S]Gαs as in Figure 4. Input equals 3% of total in vitro translation product. (B) Endogenous Gαs from rat brain lysates binds to GST-Hrs but not to GST. GST-Hrs and GST immobilized on glutathione beads were incubated with rat brain lysates (∼5 mg). Bound proteins were immunoblotted with anti-Gαs IgG. Input equals 3% of total brain lysate. (C) Myc-Hrs coimmunoprecipitates with Gαs-GFP (lane 4). Lysates (lanes 1 and 2) from HEK293 cells transfected with Gαs-GFP or GFP together with myc-Hrs were immunoprecipitated with anti-GFP, followed by immunoblotting with anti-myc and anti-GFP antibodies. (D) Gαs and Hrs are found in approximately equal amounts in both membrane (P100, lane 2) and cytosolic (S100, lane 1) fractions. Gαs coimmunoprecipitates with myc-Hrs predominantly (>95%) from membrane fractions (lane 4, bottom). Very little Gαs is coprecipitated with myc-Hrs from the cytosolic fraction (lane 3, bottom). Cytosolic (S100, lane 1) and membrane (P100, lane 2) fractions prepared from HEK293 cells transfected with Gαs and myc-Hrs were immunoprecipitated with anti-myc (myc, lanes 3 and 4) or control (ctrl, lanes 5 and 6) mouse IgGs, followed by immunoblotting with anti-Gαs and anti-myc antibodies.

Article Snippet: Specimens were analyzed using a Zeiss Axiophot equipped with a Hamamatsu Orca ER charge-coupled device (CCD) or by deconvolution microscopy by using an Applied Precision (Issaquah, WA) Delta Vision imaging system coupled to an S100 fluorescence microscope (Carl Zeiss, Thornwood, NY).

Techniques: In Vitro, Labeling, Incubation, Transfection, Immunoprecipitation, Western Blot