Journal: Biomolecules

Article Title: Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin

doi: 10.3390/biom12111698

Figure Lengend Snippet: Interaction of reduced monomer (Rec), thiol oxidation mimicking mutant (Rec-C39D), or disulfide dimer (dRec) of recoverin with caveolin-1 fragments. ( A , B ) The results of pull-down assay of Rec, Rec-C39D, or dRec (1.7 mg/mL) with affinity resins containing immobilized Cav or CavE in 20 mM Tris-HCl buffer (pH 7.5), 100 mM NaCl, 2 mM MgCl 2 , in the absence ( A ) or the presence ( B ) of calcium (1 mM CaCl 2 or 2 mM EGTA) at 25 °C. Error bars represent the weight fractions of the bound recoverin forms (in relative units, RU) determined from at least three independent experiments. *— p < 0.05. ( C – H ) Kinetics of the interaction between Rec ( C , D ), dRec ( E , F ), or Rec-C39D ( G , H ) (2.5 μM to 30 μM) with immobilized Cav or CavE in 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 2 mM DTT (except for dRec studies), 0.05% TWEEN20 in the absence (“Ca 2+ -free”) or in the presence (“Ca 2+ -loaded”) of calcium (1 mM CaCl 2 or 1 mM EDTA), determined by SPR spectroscopy at 25 °C. Blue and red sensorgrams represent experimental data, while black curves are theoretical fits calculated according to the “heterogeneous ligand” model.

Article Snippet: Rabbit monoclonal antibodies against caveolin-1 and phosphorylated caveolin-1 and mouse monoclonal antibodies against recoverin were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Mutagenesis, Pull Down Assay, Spectroscopy

Journal: Biomolecules

Article Title: Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin

doi: 10.3390/biom12111698

Figure Lengend Snippet: Parameters of the heterogeneous ligand model, describing the SPR data on the kinetics of interaction between reduced monomer (Rec), disulfide dimer (dRec), or thiol oxidation mimicking mutant (Rec-C39D) of recoverin with caveolin-1 fragments at 25 °C.

Article Snippet: Rabbit monoclonal antibodies against caveolin-1 and phosphorylated caveolin-1 and mouse monoclonal antibodies against recoverin were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Mutagenesis

Journal: Biomolecules

Article Title: Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin

doi: 10.3390/biom12111698

Figure Lengend Snippet: Disulfide dimerization of recoverin in vitro. ( A ) Kinetics of dRec formation during dialysis of 50 µM reduced recoverin against 10 mM Tris-HCl buffer (pH 7.5), 100 mM NaCl, 1 mM EGTA, containing 1 mM H 2 O 2 for 24 h at 4 °C, with or without (control) preincubation in the presence of 100 µM Cav or 100 µM CavE. ( B ) Disulfide dimerization of 50 µM reduced recoverin during dialysis (24 h, 4 °C) against 10 mM Tris-HCl buffer (pH 7.5), 100 mM NaCl, containing indicated concentrations of H 2 O 2 in the presence of 1 mM EGTA (“control”) or 4-fold molar excess of ZnCl 2 (“zinc”). Weight fractions of dRec were determined by densitometric analysis of SDS-PAGE data from at least three independent experiments and plotted against time ( A ) or H 2 O 2 concentrations ( B ).

Article Snippet: Rabbit monoclonal antibodies against caveolin-1 and phosphorylated caveolin-1 and mouse monoclonal antibodies against recoverin were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: In Vitro, Control, SDS Page

Journal: Biomolecules

Article Title: Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin

doi: 10.3390/biom12111698

Figure Lengend Snippet: Disulfide dimerization of recoverin and tyrosine (Y14) phosphorylation of caveolin-1 in MDCK-Rec cells under oxidative stress conditions. ( A ) Cells were incubated with indicated concentrations of H 2 O 2 in the presence of 0.5 mM vanadate for 10 min and their lysates were subjected to non-reducing Western blotting using antibodies against recoverin (upper panel) or P-caveolin-1 (lower panel). The positions of monomer (“Rec”), disulfide dimer (“dRec”), and disulfide aggregates (“nRec”) of recoverin, as well as P-caveolin-1 (“pCav”) are indicated by arrows. ( B , C ) Weight fractions of dRec ( B ) and pCav ( C ) estimated from Western blotting data from at least three independent experiments. *— p < 0.05 as compared to the data obtained for untreated cells.

Article Snippet: Rabbit monoclonal antibodies against caveolin-1 and phosphorylated caveolin-1 and mouse monoclonal antibodies against recoverin were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, Incubation, Western Blot

Journal: Biomolecules

Article Title: Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin

doi: 10.3390/biom12111698

Figure Lengend Snippet: Localization of caveolin-1, P-caveolin-1, and recoverin in MDCK-Rec cells under oxidative stress conditions. Caveolin-1 and P-caveolin-1 are visualized by immunocytochemical analysis using rabbit monoclonal antibodies and goat anti-rabbit Alexa Fluor 555-conjugated IgG (red). Recoverin is visualized using mouse polyclonal antibodies and goat anti-mouse Alexa Fluor 488-conjugated IgG (green). Cell nuclei are stained with DAPI (blue). ( A ) Normal conditions. White arrow indicates the area of caveolin-1 localization in the Golgi complex. ( B ) Oxidative stress conditions (10 mM H 2 O 2 ). White arrows indicate recruitment of recoverin to the plasma membrane. Red arrows point to the sites of co-localization of recoverin with P-caveolin-1. ( C ) Oxidative stress against the background of the calcium depletion conditions (5 μM BAPTA-AM). Red arrows point to the sites of co-localization of recoverin with P-caveolin-1. The insets with higher magnification (in the middle) demonstrate areas in the cytoplasm with co-localization of recoverin and non-phosphorylated caveolin-1 (indicated by white arrowheads).

Article Snippet: Rabbit monoclonal antibodies against caveolin-1 and phosphorylated caveolin-1 and mouse monoclonal antibodies against recoverin were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Bioprocessing, Staining, Clinical Proteomics, Membrane

Journal: Biomolecules

Article Title: Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin

doi: 10.3390/biom12111698

Figure Lengend Snippet: The hypothetical function of caveolin-1 complex with Rec/dRec in photoreceptors. Under normal light conditions (low calcium), recoverin forms a complex with caveolin-1 in DRMs of OS, which can be attenuated by tyrosine (Y14) phosphorylation, enabling translocation of recoverin to IS. In oxidative stress, the increased zinc concentration induces the formation of dRec, which retains in OS due to increased affinity to P-caveolin-1. The ability of dRec to constitutively inhibit rhodopsin kinase (GRK1) can slow down rhodopsin desensitization, promote oxidative stress, and induce apoptosis.

Article Snippet: Rabbit monoclonal antibodies against caveolin-1 and phosphorylated caveolin-1 and mouse monoclonal antibodies against recoverin were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, Translocation Assay, Concentration Assay