Na k atpase α

Manufactured by Santa Cruz Biotechnology

Na+/K+-ATPase α is a protein found in the cell membrane that functions as an ion pump. It is responsible for maintaining the electrochemical gradient of sodium and potassium ions across the cell membrane, which is essential for various cellular processes.

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5 protocols using na k atpase α

Antibody Sources for Western Blot

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Primary antibodies were obtained from the following sources: Na⁺/K⁺ ATPase‐α, calnexin, calpain 2, CDK2, cyclin B1, cyclin E, ERK2, lamin B, HA tag, p21, p27^Kip1, p57^Kip2, β‐tubulin from Santa Cruz Biotechnology (Santa Cruz, CA), β‐actin from Chemicon International (Billerica, MA), phospho‐AKT (Ser473), AKT, phosphorylated ERK1/2 (T202/Y204), and Lamp1 from Cell Signaling Technology (Danvers, MA) and Cathepsin B from R&D Systems (Mississauga, ON, Canada). Mouse and rabbit horseradish peroxidase antibodies were purchased from Amersham Biosciences (Pittsburg, PA), goat horseradish peroxidase antibody from Santa Cruz, and alkaline phosphatase‐conjugated antibodies were purchased from Promega (Madison, WI).

Bian B., Mongrain S., Cagnol S., Langlois M., Boulanger J., Bernatchez G., Carrier J.C., Boudreau F, & Rivard N. (2015). Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis. Molecular Carcinogenesis, 55(5), 671-687.

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Comprehensive Protein Expression Analysis

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Tissue or cell lysates were prepared by homogenization in T-PER or M-PER (Thermo Fisher Scientific). Protein lysates were subjected to SDS–polyacrylamide gel electrophoresis and probed with the following primary antibodies: PRSS8 (BD Biosciences), EGF (Santa Cruz), EGFR (Cell Signaling Technology), p-EGFR (Tyr1068, Cell Signaling Technology), Akt-1 (Santa Cruz), p-Akt (Ser473, Cell Signaling Technology), Erk1/2 (p44/42 MAPK, Cell Signaling Technology), p-Erk1/2 (Cell Signaling Technology), β-actin (Cell Signaling Technology), and Na + /K + -ATPase α (Santa Cruz). For the detection of EGFR and p-EGFR, 3%–8% Tris–acetate gels (Thermo Fisher Scientific) were used, and for others, 4%–20% Tris–Glycine gels (Thermo Fisher Scientific) were used. At least three technical replicates were performed for all blots. Full-length blots are presented in the Supplementary files.

Ishii T., Miyasato Y., Ichijo M., Uchimura K, & Furuya F. (2023). Membrane protease prostasin promotes insulin secretion by regulating the epidermal growth factor receptor pathway. Scientific Reports, 13, 9086.

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Western Blot Antibody Panel

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Western blotting antibodies were: ABCC4 (rat monoclonal M4I-10; Enzo Life Sciences, Waterloo, NSW; 1:1000), α-tubulin (mouse monoclonal DM1A; Abcam; 1:3000), total actin (rabbit polyclonal A2066; Sigma-Aldrich; 1:2000), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; mouse monoclonal G-9, sc365062; Santa Cruz Biotechnology, Dallas, TX; 1:5000), Na+/K+-ATPase α (rabbit polyclonal H-300, sc-28800; Santa Cruz Biotechnology; 1:1000), peroxidase-conjugated goat anti-rat IgG (VWR International, Murarrie, QLD; 1:10000) and sheep anti-mouse horseradish peroxidase (GE Healthcare, Rydalmere, NSW; 1:50000).

Murray J., Valli E., Yu D.M., Truong A.M., Gifford A.J., Eden G.L., Gamble L.D., Hanssen K.M., Flemming C.L., Tan A., Tivnan A., Allan S., Saletta F., Cheung L., Ruhle M., Schuetz J.D., Henderson M.J., Byrne J.A., Norris M.D., Haber M, & Fletcher J.I. (2017). Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumor growth and sensitizes to irinotecan in vivo. European journal of cancer (Oxford, England : 1990), 83, 132-141.

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Modulation of Dendritic Cell Activation

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The culture medium consisted of RPMI 1640 (Gibco-BRL, Life Technologies Corporation, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS; Hyclone, Thermo Fisher Scientific Inc, Waltham, MA, USA). Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were purchased from R&D Systems (Minneapolis, MN, USA). Mitogen-activated protein kinase (MAPK) inhibitors (SB203580 for p38, PD98059 for ERK and SP600125 for JNK) were purchased from Calbiochem (Merck KGaA, Darmstadt, Germany). The antibodies (Abs) recognizing CD80, CD86, CD83 and HLA-DR were purchased from BD Pharmingen (BD Biosciences, Franklin Lakes, NJ, USA). Antibodies recognizing total or phosphorylated c-Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK) were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or Cell Signaling (Beverly, MA, USA). Antibodies against p65, p50, p52, RelB, c-Rel, Na⁺/K⁺-ATPase α, Toll-like receptor-3 (TLR-3) and upstream stimulatory factor (USF)-2 were purchased from Santa Cruz. Anti-CCR7, anti-IL-12p40 and anti-Gal-9 Abs and chemoattractants, including CCL19 and CCL21, were purchased from R & D. Anti-Tim-3 neutralizing Abs (LEAF™ purified anti-human Tim-3 antibody) were purchased from BioLegend (San Diego, CA, USA).

Hsu Y.L., Wang M.Y., Ho L.J., Huang C.Y, & Lai J.H. (2015). Up-regulation of galectin-9 induces cell migration in human dendritic cells infected with dengue virus. Journal of Cellular and Molecular Medicine, 19(5), 1065-1076.

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Muscle Protein Content Analysis

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Muscle protein content was assessed by Western blot as previously described (Martinez et al. 2016 (link), Rodriguez et al. 2012 (link), Vázquez-Medina et al. 2013 (link)). Membranes were incubated with primary antibodies against AMPKα (1:500, Cell Signaling Technology, Danvers, MA), phospho-AMPKα (Thr172) (1:350, Cell Signaling Technology), insulin receptor-β (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), phospho-IGF-I receptor-β (1:500, Cell Signaling Technology), and Na+/K+-ATPase-α (1:1000, Santa Cruz Biotechnology). Membranes were washed and incubated with secondary antibodies IR Dye 680RD donkey anti-mouse, IR Dye 680 RD donkey anti- rabbit, or IR Dye 800CW donkey anti-rabbit (1:20000). Proteins were then visualized using the LI-COR Odyssey Infrared Imaging System and quantified with ImageJ (NIH, Bethesda, MD). In addition consistency in loading equivalent amounts of total protein was confirmed and normalized by correcting for the densitometry values of Ponceau S staining (Gilda & Gomes 2013 (link)).

Vazquez-Anaya G., Martinez B., Soñanez-Organis J.G., Nakano D., Nishiyama A, & Ortiz R.M. (2016). Exogenous Thyroxine Improves Glucose Intolerance in Insulin Resistant Rats. The Journal of endocrinology, 232(3), 501-511.

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