rabbit anti na k atpase alpha1 subunit  (Novus Biologicals)

Journal: Frontiers in Oncology

Article Title: Displacement of Native FXYD Protein From Na + /K + -ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity

doi: 10.3389/fonc.2022.859216

Figure Lengend Snippet: Amino acid sequences of FXYD3 and its derivatives. (A) Wild-type (WT) FXYD3. Cys residues mutated to Ser in full-length recombinant protein and the FXYD motif in the extracellular domain are shown in red font and are underlined. A Cys residue (indicated by *) in the cytoplasmic domain near the inner membrane leaflet and bracketed by basic amino acids (here Lys) is obligatory for susceptibility to glutathionylation of members of the FXYD protein family. Such susceptibility is critical for a FXYD protein, including FXYD3, to counter glutathionylation of the β1 Na + /K + -ATPase subunit. (B) Amino acid sequence of FXYD3-pep SKSK. Ser residues (indicated by *) that replaced Cys residues in the corresponding sites in WT FXYD3 are indicated in red font. (C) Amino acid sequence of FXYD3-pep CKCK that has Cys residues (indicated by *) of WT FXYD3 retained. (D) Immunoblots for GSH indicating glutathionylation of FXYD3-pep CKCK but not FXYD3-pep SKSK with exposure to GSSG or GSH/H 2 O 2 . (E) Reversibility of FXYD3-pep CKCK glutathionylation with exposure to DTT. (F) Immunoblot for GSH of cell lysate immunoprecipitated for the β1 Na + /K + -ATPase subunit after exposure to Dox. Pre-exposure to FXYD3-pep SKSK augments Dox-induced β1 Na + /K + -ATPase subunit glutathionylation.

Article Snippet: After solubilization in RIPA lysis buffer, protein extracts (0.5–1 mg) from cells were precleared and incubated overnight at 4°C with anti-α 1 subunit Na + -K + ATPase (D4Y7E; Cell Signaling Technology, USA), anti-FXYD3 (ab205534; Abcam, UK), or anti-β1 subunit (D6U8Q; Cell Signaling Technology, USA) Na + /K + -ATPase antibodies followed by precipitation for 2 h at 4°C with protein A/G plus agarose-coated beads (Abcam, UK).

Techniques: Recombinant, Residue, Membrane, Sequencing, Western Blot, Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: Displacement of Native FXYD Protein From Na + /K + -ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity

doi: 10.3389/fonc.2022.859216

Figure Lengend Snippet: Distribution of FXYD3-pep SKSK and its ability to displace FXYD3 from the α1 Na + /K + -ATPase subunit. (A) FXYD3 and Na + /K + -ATPase α1 subunit expression in MCF-7 and MDA-MB-468 breast cancer cells and in BxPC-3 and Panc-1 pancreatic cancer cells. Expression is normalized to expression in human non-cancer MCF-10A cells; GAPDH was the internal loading control. (B) Immunofluorescence showing distribution of TRITC-labeled FXYD3-pep SKSK in BxPC-3 cells. Cells were exposed to 1 µM TRITC-tagged FXYD3-pep SKSK (TRITC-SKSK) (red) for 2 h and fluorescence microscopy performed 24 h after the peptide was washed off. DAPI was used to counterstain the nucleus (blue). One of 5 similar experiments is shown. Fluorescence is predominantly peri-nuclear. Scales are shown in the middle panel. (C) Immunoblot (IB) of α1 Na + /K + -ATPase subunit with WT FXYD3 immunoprecipitant in lysate of BXPC-3 with and without exposure of the cells to 1 μM FXYD3-pep SKSK for 2 h before lysis. (D) Immunoblot of WT FXYD3 with the α1 subunit immunoprecipitant in the lysate from the cells. C, control; TL, total lysate; non-immune IgG (IgG), negative control for IP. The efficiency of the Co-Ip can be estimated by the comparison of β1 subunit expression in the initial total lysate and the unbound supernatant after IP in BxPC-3 cells (data not shown). Approximate binding efficiency was ~90%.

Article Snippet: After solubilization in RIPA lysis buffer, protein extracts (0.5–1 mg) from cells were precleared and incubated overnight at 4°C with anti-α 1 subunit Na + -K + ATPase (D4Y7E; Cell Signaling Technology, USA), anti-FXYD3 (ab205534; Abcam, UK), or anti-β1 subunit (D6U8Q; Cell Signaling Technology, USA) Na + /K + -ATPase antibodies followed by precipitation for 2 h at 4°C with protein A/G plus agarose-coated beads (Abcam, UK).

Techniques: Expressing, Control, Immunofluorescence, Labeling, Fluorescence, Microscopy, Western Blot, Lysis, Negative Control, Co-Immunoprecipitation Assay, Comparison, Binding Assay

Journal: Frontiers in Oncology

Article Title: Displacement of Native FXYD Protein From Na + /K + -ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity

doi: 10.3389/fonc.2022.859216

Figure Lengend Snippet: Plasmalemmal Na + /K + -ATPase as a potential target for FXYD3-pep SKSK. (A) MCF-7 cell survival after a 48-h exposure to ouabain in concentrations indicated, with or without co-exposure to 1 μM doxorubicin (Dox). * indicates difference in additive effect of ouabain and Dox at 25 or 50 nM ouabain vs. 70–150 nM ouabain. N = 5 for each ouabain concentration. (B) Electrogenic Na + /K + -ATPase pump currents (I p ) of MCF-7 cells that do, and MDA468 cells that do not, overexpress Na + /K + -ATPase. The trace of membrane current (Im) was recorded in an MCF-7 cell before and after Na + -K + pump activity was eliminated with exposure to K + -free extracellular solution. The inward shift to a near-zero holding current in K + -free solution identifies I p . Currents were sampled with an electronic cursor after electrical noise caused by extracellular solution change had subsided.

Article Snippet: After solubilization in RIPA lysis buffer, protein extracts (0.5–1 mg) from cells were precleared and incubated overnight at 4°C with anti-α 1 subunit Na + -K + ATPase (D4Y7E; Cell Signaling Technology, USA), anti-FXYD3 (ab205534; Abcam, UK), or anti-β1 subunit (D6U8Q; Cell Signaling Technology, USA) Na + /K + -ATPase antibodies followed by precipitation for 2 h at 4°C with protein A/G plus agarose-coated beads (Abcam, UK).

Techniques: Concentration Assay, Membrane, Activity Assay