The VSMCs were cultured in 60‐mm dishes and they were lysed at each point in time. Subcellular proteins were fractionated by the Subcellular Protein Fractionation Kit (78840, Thermo Fisher) according to the manufacturer's instructions. The extractions were separated by SDS‐PAGE and AT1R levels were analyzed by Western blot with use of rabbit anti‐AT1R antibodies (1:1000, ab124734, Abcam, UK). Rabbit anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and rabbit anti‐Na+/K+ ATPase from Abcam (UK) were detected as cytoplasm or membrane loading control, respectively.
Rabbit anti na k atpase
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti‐Na+/K+ ATPase is a primary antibody that specifically recognizes the sodium‐potassium adenosine triphosphatase (Na+/K+ ATPase) enzyme. The Na+/K+ ATPase is a membrane‐bound protein responsible for the active transport of sodium and potassium ions across the cell membrane, maintaining the electrochemical gradient.
Automatically generated - may contain errors
Lab products found in correlation
Subcellular protein fractionation kit, by Thermo Fisher Scientific (1 mentions)
Ab124734, by Abcam (1 mentions)
Image studio, by LI COR (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Irdye 680rd goat anti mouse, by LI COR (1 mentions)
Neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Proteintech (1 mentions)
Rabbit anti nis, by Abcam (1 mentions)
Rabbit anti cd97, by Abcam (1 mentions)
Rabbit anti gapdh, by Proteintech (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by ZSGB-BIO (1 mentions)
High capacity neutravidin agarose, by Thermo Fisher Scientific (1 mentions)
Proteiniso gst resin, by Transgene (1 mentions)
Rabbit anti flag, by Proteintech (1 mentions)
Goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
5 protocols using rabbit anti na k atpase
1
Subcellular Fractionation and AT1R Analysis
2
Cell Surface Biotinylation of Mouse Brain Slices
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Cell surface biotinylation of mouse brain slices was conducted as published [62 (link)]. After euthanization, mouse brains were freshly isolated and sliced into 1 mm coronal sections. Brain slices were incubated in 1 mg/ml sulfo-NHS-SS-biotin (ThermoFisher) in pre-chilled oxygenated aCSF on ice for 30 min and then washed twice with 50 mM glycine and three times with 1 mg/ml BSA in pre-chilled oxygenated aCSF. Hippocampus was dissected from slices, extracted and the biotinylated proteins purified using NeutrAvidin beads (ThermoFisher) and processed for Western blot with following antibodies: rabbit anti CLC-3 (Rabbit, 1:1000, Alomone, ACL-001), Rabbit anti-Na-K ATPase (1:10,000, Abcam, ab76020). Blots were developed with goat anti-mouse IRDye 680RD and goat anti-rabbit IRDye 800CW (1:5000, LI-COR) and Odyssey CLx imager (LI-COR) and quantitated using Image Studio (LI-COR).
3
Western Blot Analysis of Protein Targets
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Western blot analysis was performed using standard protocols as described previously (26 (link), 27 (link)). All protein extracts were denatured in a boiling water bath (95°C) for 5 min except for NIS, which was heated at 37°C for 30 min. The antibodies and their dilutions used were as follows: rabbit anti-NIS (1:200; Abcam, UK); rabbit anti-CD97 (1:1,000; Abcam, UK); rabbit anti-GAPDH (1:5,000; Proteintech, China); rabbit anti-Na+/K+-ATPase (1:1,000; Abcam, UK); rabbit anti-cAMP (1:1,000; Abcam, UK); rabbit anti-pCREB (Ser133) (1:1,000; Abcam, UK); mouse anti-TSHR (1:1,000; Abcam, UK); peroxidase conjugated goat anti-rabbit IgG (1:10,000; ZSGB-BIO, China); peroxidase conjugated goat anti-mouse IgG (1:10,000; Proteintech, USA). The quantitative analysis of band intensity was performed by ImageJ software.
4
Immunofluorescence Analysis of Stomach Cells
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Stomach sections were processed for single- and double-labeling immunofluorescence. Sections were deparaffinized, rehydrated, and incubated for 1 h in a humid chamber at room temperature (RT) with appropriate normal serum (5% normal goat or donkey serum) and 1% BSA diluted in PBS (phosphate buffered saline, 0.01 M pH 7.4) to reduce the nonspecific binding of the secondary antibodies. The sections were then incubated at 4 °C in a humid chamber for 24 h with the primary antibody rabbit anti-Na+K+-ATPase (Abcam, Cambridge, UK) diluted to 1:200. After washing, the sections were incubated at RT for 1 h with the goat anti-rabbit Alexa Fluor® 488.
For the EECs, rat anti-somatostatin (Enzo Life Sciences, New York, NY, USA) diluted to 1:300, was used in association with mouse anti-ghrelin (Acris/OriGene, Herford, Germany) diluted to 1:300 in PBS. The sections were incubated at 4 °C overnight. After washing, the sections were incubated at RT for 1 h with donkey anti-rat Alexa Fluor® 488 and donkey anti-mouse Alexa Fluor® 594 secondary antibody (Invitrogen, CA, USA) diluted in PBS and then coverslipped with buffered glycerol, pH 8.6.
For the EECs, rat anti-somatostatin (Enzo Life Sciences, New York, NY, USA) diluted to 1:300, was used in association with mouse anti-ghrelin (Acris/OriGene, Herford, Germany) diluted to 1:300 in PBS. The sections were incubated at 4 °C overnight. After washing, the sections were incubated at RT for 1 h with donkey anti-rat Alexa Fluor® 488 and donkey anti-mouse Alexa Fluor® 594 secondary antibody (Invitrogen, CA, USA) diluted in PBS and then coverslipped with buffered glycerol, pH 8.6.
5
Biochemical Reagents for Cell Signaling
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Rabbit anti-TRPV1 (Alomone laboratory, catalog no. ACC-030), mouse anti-Kvβ1 (Santa Cruz, catalog no. sc-373986), rabbit anti-Na+/K+ ATPase (Abcam, catalog no. ab76020), mouse anti-GST (Abclonal, catalog no. AE001), mouse anti-β-actin (Transgen Biotech, catalog no. 6609-1), mouse anti-FLAG (Sigma, catalog no. F3165), mouse anti-FLAG (MBL, catalog no. M185-3L), rabbit anti-FLAG (Proteintech, catalog no. 20543-1-AP), mouse anti-HA (MBL, catalog no. M180-3), rabbit anti-HA (Earthox, catalog no. E02218002), mouse anti-GFP (Tianjin Sungene Biotech, catalog no. KM8009), rabbit anti-GFP (WB: Solarbio, catalog no. B1025F), anti-FLAG affinity gel (Bimake, catalog no. B23100), anti-HA affinity gel (Bimake, catalog no. B23301), ProteinIso GST resin (Transgen Biotech, catalog no. DP201-01), goat anti-mouse IgG (H + L) (Jackson Immunoresearch, catalog no. 115-035-003), and goat anti-rabbit IgG (H + L) (Jackson Immunoresearch, catalog no. 111-005-003) were purchased from the indicated manufactures. The EZ-Link Sulfo-NHS-LC-Biotin (catalog no. 21335) and high-capacity NeutrAvidin-agarose (catalog no. 29202) were ordered from Thermo Fisher Scientific.
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