Journal: Biological Research

Article Title: Inhibition of forward and reverse transport of Ca 2+ via Na + /Ca 2+ exchangers (NCX) prevents sperm capacitation

doi: 10.1186/s40659-024-00535-9

Figure Lengend Snippet: Identification of NCX isoforms. Identification of NCX1, NCX2 and NCX3 in porcine sperm. Representative immunoblots using sperm samples from different boars with A ) anti-NCX1, ( B ) anti-NCX2 and ( C ) anti-NCX3 antibodies, and their respective blocking peptides

Article Snippet: To block nonspecific binding sites, samples were incubated with a blocking solution consisting of 5% BSA in PBS at room temperature for 1 h. Subsequently, sperm were incubated with primary NCX antibodies, either NCX1, NCX2 or NCX3 (Alomone Labs), diluted 1:100 (v: v) at room temperature for 1 h. After washing five times with PBS (5 min per wash), samples were incubated with a secondary anti-rabbit antibody Alexa Fluor Plus 488 (ref. A32731, Invitrogen, Waltham, MA, USA) diluted 1:200 (v: v) in blocking solution at room temperature for 1 h. Samples were again washed five times with PBS (5 min per wash), air dried and mounted with 10 μL of ProLongTM Glass Antifade Mountant with NucBlue (Hoechst 33342; ref. P36985, Invitrogen) in the dark.

Techniques: Western Blot, Blocking Assay

Journal: Biological Research

Article Title: Inhibition of forward and reverse transport of Ca 2+ via Na + /Ca 2+ exchangers (NCX) prevents sperm capacitation

doi: 10.1186/s40659-024-00535-9

Figure Lengend Snippet: Immunolocalization of NCX1 isoform. Localization of NCX1 in the plasma membrane of porcine sperm ( A – C ), and after the peptide competition assay ( D – F ). NCX1 appears stained in green (Alexa Fluor 488) and nuclei in blue (DAPI; 4′6′-diamidion-2-phenylindole). Scale bar: 15 μm

Article Snippet: To block nonspecific binding sites, samples were incubated with a blocking solution consisting of 5% BSA in PBS at room temperature for 1 h. Subsequently, sperm were incubated with primary NCX antibodies, either NCX1, NCX2 or NCX3 (Alomone Labs), diluted 1:100 (v: v) at room temperature for 1 h. After washing five times with PBS (5 min per wash), samples were incubated with a secondary anti-rabbit antibody Alexa Fluor Plus 488 (ref. A32731, Invitrogen, Waltham, MA, USA) diluted 1:200 (v: v) in blocking solution at room temperature for 1 h. Samples were again washed five times with PBS (5 min per wash), air dried and mounted with 10 μL of ProLongTM Glass Antifade Mountant with NucBlue (Hoechst 33342; ref. P36985, Invitrogen) in the dark.

Techniques: Clinical Proteomics, Membrane, Competitive Binding Assay, Staining, Immunofluorescence

Journal: Frontiers in Physiology

Article Title: Pharmacological Preconditioning Using Diazoxide Regulates Store-Operated Ca 2 + Channels in Adult Rat Cardiomyocytes

doi: 10.3389/fphys.2019.01589

Figure Lengend Snippet: Orai1 associates with NCX1 in the adult rat heart. (A,B) Orai1 and NCX1 were immunoprecipitated from membrane fractions of control and PPC ventricles, respectively, and. immunoblotted with anti-Orai1 antibody. (C) NCX1 from membrane fractions of control and PPC ventricles was immunoblotted with anti-NCX1 antibody (input). (D) Orai1-immunoprecipitated proteins were immunoblotted with anti-NCX1 antibody.

Article Snippet: The source of antibodies was: polyclonal anti-NCX1 antibody (Proteintech Group, Inc., Rosemont, IL, United States), and polyclonal anti-Orai1 (ThermoFisher Scientific, Waltham, MA, United States).

Techniques: Immunoprecipitation, Membrane, Control

Journal: PLoS ONE

Article Title: Role of Na + /Ca 2+ Exchanger (NCX) in Modulating Postovulatory Aging of Mouse and Rat Oocytes

doi: 10.1371/journal.pone.0093446

Figure Lengend Snippet: The micrographs are laser confocal images (equatorial sections) of oocytes with NCX1 pseudo-colored red. They show the cortical distribution of NCX1 in newly-ovulated mouse oocytes (A) or oocytes cultured for 6 h in CZB medium without (B) or with (C) H 2 O 2 addition. Graphs D and E show NCX1 quantification, respectively, in mouse and rat oocytes recovered at different times post hCG injection. Graph F shows NCX1 quantification after mouse oocytes recovered 19 h post hCG were cultured for 6 h with (+N) or without (-N) 75 mM NaCl supplementation. Graph G shows NCX1 quantification in newly-ovulated mouse oocytes (0h), or oocytes cultured for 6 h in CZB medium with (6h+O) or without (6h-O) H 2 O 2 addition. Each treatment was repeated four times, with each replicate containing 7–10 oocytes. a-c: Values without a common letter above their bars differ (P<0.05).

Article Snippet: Oocytes were (i) fixed with 3.7% paraformaldehyde in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA and 4 mM MgSO 4 , pH 7.0) for at least 30 min, followed by treatment with 0.25% protease in M2 for 1 to 2 seconds to remove zona pellucida; (ii) permeabilized with 0.1% Triton X-100 in PHEM for 5 min; (iii) blocked in PHEM containing 3% BSA for 1 h; (iv) incubated overnight with mouse monoclonal anti-NCX1 (IgM, 1∶400, Abcam, Cambridge, MA) in 3% BSA in M2 at 4°C; (v) incubated for 1 h with Cy3-conjugated goat-anti-mouse IgM (1∶800, Jackson ImmunoResearch) in 3% BSA in M2.

Techniques: Cell Culture, Injection