Protein concentrations were estimated using the BCA Protein Assay Kit (Thermo Fisher Scientific). Equal quantities of protein lysates (20 μg) were separated by 10% SDS-PAGE and then electro-transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature, and then incubated overnight at 4°C with the following antibodies: Arginase1 (1: 1000, Abcam Cambridge, MA, USA), iNOS (1: 1000, Abcam), Klotho (1: 1000, Abcam), α-SMA (1: 1000, Abcam), p-p65 (1: 1000, Abcam), p65 (1: 1000, Abcam), GAPDH (1: 1000, Abcam). The following day, the membranes were washed and then incubated with the corresponding secondary antibodies (1:5000, Abcam) for 2 h at room temperature. Proteins were visualized using an ECL Substrate Reagent Kit (Thermo Fisher Scientific).
Klotho
Manufactured by Abcam
Sourced in United States, China
Klotho is a protein that functions as a co-receptor for the fibroblast growth factor (FGF) signaling pathway. It plays a role in the regulation of phosphate and vitamin D metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
α sma, by Abcam (2 mentions)
β actin, by Wuhan Servicebio Technology (2 mentions)
Catalase, by Abcam (2 mentions)
Enhanced chemiluminescence substrate, by Bio-Rad (2 mentions)
Fuji blue x ray film, by Bio-Rad (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Arginase1, by Abcam (1 mentions)
Ecl substrate reagent kit, by Thermo Fisher Scientific (1 mentions)
Phosphorylated iκb α, by Abcam (1 mentions)
Histone 3, by Cell Signaling Technology (1 mentions)
17 protocols using klotho
1
Western Blot Analysis of Protein Expression
2
Protein Extraction and Western Blotting
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Total protein was extracted from mouse kidneys and cultured cells using RIPA Lysis Buffer (Beyotime Biotechnology, Nanjing, China). Nucleoproteins were extracted using a commercial kit (Beyotime Biotechnology). Samples (20–40 µg of protein/lane) were separated on 12% (w/v) sodium dodecyl sulfate-polyacrylamide gels; the proteins were electrotransferred to 0.22-µm pore-sized polyvinylidene fluoride membranes (Millipore Sigma, Burlington, MA, USA) and immunoblotted with primary antibodies against Klotho (Abcam; 1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), NF-κB p65 (Cusabio, Wuhan, China; 1:1,000), IκB-α (Abcam; 1:5,000), phosphorylated-IκB-α (Abcam; 1:1,000), COX2 (Proteintech, Rosemont, IL, USA; 1:300), caspase-3 (Proteintech; 1:1,000), Bax (Proteintech; 1:2,000), Bcl2 (Cell Signaling Technology, Danvers, MA, USA; 1:1,000), histone 3 (Cell Signaling Technology; 1:1,000), and β-actin (Servicebio; 1:2,000). They were then incubated with HRP-conjugated secondary antibodies (Proteintech; 1:10,000). The Western blots were developed using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA); band intensities were quantified with the aid of Quantity One ver. 4.6.7 software (Bio-Rad).
3
Quantitative Renal Immunohistochemistry Analysis
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Immunohistochemistry was performed as described previously [40 (link)]. The renal tissue slides were incubated with primary antibodies against klotho (Abcam, Cambridge, MA) and LC3 I/II (Cell Signaling, Beverly, MA) and secondary horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulins (Abcam, New Territories, HK). Using light microscopy, changes in the kidneys and the positively stained areas were observed. These positive areas were visualized at a magnification of 200 ×, and the percentages of the positive areas in the whole renal areas were calculated in 3 randomly selected, nonoverlapping fields with Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD).
4
Protein Expression Analysis of Extracellular Vesicles and Renal Tissue
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Ten micrograms of EV-lysates and 50 μg of renal tissue lysates were loaded on Mini-PROTEAN TGX pre-cast electrophoresis gels (Bio-Rad, Hercules, CA, USA). Proteins were subsequently transferred on iBlot nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA) and blotted with antibodies against Klotho (Abcam, Cambridge, UK), vinculin (Sigma-Aldrich), AQP1 (Santa Cruz Biotechnology, Dallas, TX, USA), AQP2 (Santa Cruz Biotechnology), CD63 (Santa Cruz Biotechnology), and calreticulin (Cell Signaling, Danvers, MA, USA). Chemiluminescent signal was detected using the ECL substrate (Bio-Rad).
5
Western Blot Analysis of Vascular Proteins
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Aortic tissues or cell extracts containing equal amounts of total protein were resolved by 10% or 12% SDS-PAGE, then transferred to a nitrocellulose membrane. Nonspecific proteins were blocked with 5% nonfat dried milk for 1 h, then incubated with the primary antibodies for β-actin (#sc-47778; 1:3000), GAPDH (#sc-47724; 1:1000; both Santa Cruz Biotechnology), CRLR (#GTX64616; 1:300; GeneTex, Irvine, CA, USA), RAMP1 (#ab156575; 1:1000; Abcam), RAMP2 (#sc-365240; 1:100), RAMP3 (#sc-365313; 1:100; both Santa Cruz Biotechnology); sirt1 (#ab110304; 1:200), klotho (#ab203576; 1:500), RUNX2 (#ab23981; 1:1000), BMP2 (#ab14933; 1:500; all Abcam); p53 (#sc-6243; 1:100; Santa Cruz Biotechnology), and acetyl-p53 (#ab183544; 1:500), p21 (#ab109199; 1:500), p16 (#ab189034; 1:500), α-SMA (#ab5694; 1:3000), SM-22α (#ab10135; 1:1000; all Abcam); MGP (#10734-1-AP; 1:500; Proteintech, USA), AMPK (#2532; 1:1000), p-AMPK (Thr172) (#2531; 1:1000), Akt (#9272; 1:1000), p-Akt (Ser473) (#4060; 1:1000), PKA (#4782; 1:1000) or p-PKA (Thr197) (#4781; 1:1000; all Cell Signaling Technology) overnight at 4 °C, then secondary antibody (horseradish peroxidase-conjugated anti-rabbit, anti-mouse or anti-goat IgG) (Santa Cruz Biotechnology) for 1 h. The proteins were detected by enhanced chemiluminescence. Protein expression was analyzed by using ImageJ and normalized to β-actin or GAPDH expression.
6
Protein Expression Analysis of Sciatic Nerve
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The total protein of sciatic nerve tissue was extracted. First, the nucleus protein was extracted by nucleus protein extraction kit (Beyotime Bio, China). The content was detected by BCA protein detection kit (Thermo, Waltham, MA) according to the instructions. Then, the protein (30 µg/samples) with 10% SDS-PAGE was isolated and transferred to the PVDF membrane, sealed in 5% skim milk under 25°C for 1 h, and incubated with primary NF-B P65 (1:2000, Abcam Biotech, Cambridge, MA, USA), Caspase1 (1:1000, Abcam), Pro-Caspase1 (1:1000, Cusabiao, Wuhan, China), GSDMD-N (1:1000, Abcam), Klotho (1:1000, Abcam), NLRP3 (1:1000, Abcam), GAPDH (1:2500, Abcam) and H3 (1:2000, Abcam). And then, it was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime, Shanghai, China) for 1 h. After that, protein bands were detected with a ECL detection kit (Beyotime Biothech, Shanghai, China), GAPDH or H3 was taken as control.
7
Comprehensive Molecular Signaling Pathway Analysis
8
Quantitative Protein Expression Analysis
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The protein expression levels were determined by Western blotting. The samples were re-labeled for a blind analysis before the Western blot processing. Equal amounts of protein extracts were separated using sodium dodecyl sulfate (SDS)-PAGE and then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the primary antibodies for E-cadherin (#866702), vimentin (#677802), α-SMA (#904601) (Biolegend, San Diego, CA, USA), p53 (#sc-126), p16 (#sc-377412), p21 (#sc-6246), β-actin (#sc47778), GAPDH (#sc25778) (Santa Cruz Dallas, TX, USA), catalase (#ab16731), superoxide dismutase 1 (SOD1; #ab13498), Klotho (#ab203576), CD68 (#ab125212), TNF-α (#ab183218) (Abcam, Cambridge, MA, USA), Ly6G (#14-5931-82) (eBioscience, San Diego, CA, USA), Bax (#14796), Bcl-2 (#3498), cleaved Caspase 3 (#9664), and CHOP (#2895) (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). The blot signals were measured using enhanced chemiluminescence substrates (Bio-Rad) and developed onto a Fuji Blue X-ray Film. ImageJ software was used to quantify the protein expression bands.
9
Immunocytochemical Investigation of Dermal Merkel Cells
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The following primary monoclonal antibodies were used for an immunocytochemical investigation: type I collagen (1:150, Abcam), elastin (1:50, Abcam), AP-1 (1:200, Sigma), Klotho (1:250, Abcam), MTH-1 (1:1000, Abcam), melatonin receptor 1A (1:200, Abcam), melatonin receptor 1B (1:100, Abcam), clock (1:100, Abcam), cytokeratin 20 (1:150, Dako). The secondary antibodies conjugated with Alexa Fluor 488 and 647 (1:1000, Abcam) were used for immunofluorescent microscopy. Confocal microscopy was carried out using an Olympus FV 1000 microscope (Japan).
Since the investigation was concerned with unique dermal Merkel cells, to confirm the existence of a subpopulation of these cells in the culture, we used a special marker cytokeratin-20. We applied the method of double labeling using antibodies to cytokeratin-20 (green fluorescence) and antibodies to Klotho (red fluorescence). The area of expression was calculated in relation to Merkel cells that were immunopositive to CK-20 cells.
Since the investigation was concerned with unique dermal Merkel cells, to confirm the existence of a subpopulation of these cells in the culture, we used a special marker cytokeratin-20. We applied the method of double labeling using antibodies to cytokeratin-20 (green fluorescence) and antibodies to Klotho (red fluorescence). The area of expression was calculated in relation to Merkel cells that were immunopositive to CK-20 cells.
10
Protein Extraction and Western Blot Analysis
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Total protein was extracted from renal cortices and HK2 cells using RIPA Lysis Buffer (KeyGEN BioTECH, China), and protein concentrations were estimated by bicinchonininc acid (BCA) assay (Takara Biotechnology). Equal amounts of protein were fractionated by 10%–12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Hercules, California, USA) and then electrotransferred onto polyvinylidene fluoride membranes (Merck Millipore, MA, USA), which were blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% skim milk before being incubated with primary antibodies against Klotho (1:500 dilution, Abcam), Egr-1 (1:500 dilution, Santa Cruz Biotechnology), E-cadherin (1:500 dilution; BD, USA), α-SMA (1:500 dilution; Boster, USA), FN (1:1000 dilution; R&D, USA), ERK1/2 (1:1000 dilution; CST, USA), phosphorylated ERK1/2 (p-ERK1/2) (1:500 dilution, CST) and β-actin (1:1000 dilution, ZSGB-BIO, China) overnight. The membranes were then incubated with a fluorescent secondary antibody (1:10 000 dilution; LI-COR, USA) for 1 hour. The signal was visualized using an Odyssey Infrared Imaging System (LI-COR) and quantified using Quantity One V.4.4.0 software (Bio-Rad Laboratories).
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