Islet and cell proteins were extracted with RIPA. Extracted proteins were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Bio-rad Heidemannstraße, Germany), which were blocked with 5% milk and then proved overnight with anti-FGF21 (Abcam, Cambridge, UK), anti-phospho-FRS, 2-anti-FRS2, anti-phospho-ERK1/2, anti-ERK1/2 (Cell Signaling Technology, Boston, MA), or anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies at room temperature (RT). Horseradish peroxide-conjugated secondary antibodies were incubated at RT with membranes for 2 h after washing with phosphate buffered saline with 0.1% Tween-20. Labeled protein bands were visualized on autoradiography films (Fuji Film, Tokyo, Japan) following application of ECL detection reagent (GE healthcare, Chicago, IL). Protein bands were quantitated in Image software (National Institutes of Health, Bethesda, MD) and normalized to β-actin. The primary and secondary antibodies used are listed in Table 2 .
Anti fgf21
Manufactured by Abcam
Sourced in United Kingdom
Anti-FGF21 is a laboratory antibody product used for the detection and quantification of Fibroblast Growth Factor 21 (FGF21) in various biological samples. FGF21 is a metabolic regulator that plays a role in glucose and lipid homeostasis. This antibody can be used in techniques such as Western blotting, ELISA, and immunohistochemistry to measure FGF21 levels.
Automatically generated - may contain errors
Lab products found in correlation
Autoradiography film, by Fujifilm (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Ecl detection reagent, by GE Healthcare (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Anti sirt1, by Abcam (2 mentions)
Anti β klotho, by Abcam (2 mentions)
Citrate buffer, by Abcam (2 mentions)
Chicken anti vimentin, by Merck Group (2 mentions)
Donkey anti rabbit af594 and donkey anti chicken af647, by Jackson ImmunoResearch (2 mentions)
Goat anti chicken af488, by Jackson ImmunoResearch (2 mentions)
C fos antibody, by Cell Signaling Technology (2 mentions)
Bx61 light microscope, by Olympus (2 mentions)
Cryostat, by Leica (2 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (2 mentions)
Glucosamine hydrochloride, by Merck Group (1 mentions)
Reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
10 protocols using anti fgf21
1
Western Blot Analysis of Islet Proteins
2
Molecular Signaling Analysis in FBS-Treated Cells
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Fetal bovine serum (FBS) was purchased from Cytiva (Marlborough, MA, USA). Reverse transcriptase and SYBR green reagents were obtained from ThermoFisher (ThermoFisher Scientific, Waltham, MA, USA). Antibodies were obtained from different sources: anti-β-actin (Novus Cat. # NB600-501; St. Louis, MO, USA), anti-FGF21 (abcam Cat. #ab171941), anti-phospho-Akt (Ser473) (Cell Signaling, Cat. # 9271; Danvers, MA, USA), anti-Akt (Cell Signaling, Cat. # 4691), anti-phospho-CREB (87G3) (Cell Signaling, Cat. # 9197), anti-CREB (48H2) (Cell Signaling Cat. # 9197), anti-phospho-JNK (Thr183/Thr185) (Cell Signaling, Cat. # 9251), anti-JNK (Cell Signaling, Cat. # 9258), anti-phospho-p38 (Thr182) (Santa Cruz Cat. # sc-7973), anti-p38 (Cell Signaling, Cat. # 9212), anti-phospho-NF-κB p65 (S536) (R&D Systems, Cat. # KCB7226; Minneapolis, MN, USA), and anti-NF-κB p65 (Cell Signaling, Cat. # 8242). The materials for the animal diets were obtained from Research Diets Inc. (New Brunswick, NJ, USA). Glucosamine hydrochloride (Cat. 66842), along with all other unspecified chemicals and reagents utilized in this project, was procured from Sigma-Aldrich (St. Louis, MO, USA).
3
Protein Expression Analysis in Kidney
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Analysis was performed to measure the protein levels in kidney tissue and HK-2 cells. Protein in total cell lysates was extracted and quantified by BCA Protein Assay. Protein was separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules). After blocking the membrane in a TBST solution containing 10% (w/v) non-fat milk for 1.5 h at room temperature, different proteins were probed with corresponding antibodies overnight at 4°C. The following antibodies were used: anti-FGF21 (1:1000, Abcam), anti-SIRT1 (1:1000, Abcam), anti-TIM-1 (1:20,000, Abcam), anti-Bax (1:10,000, Abcam), anti-cleaved caspase-3 (1:1000, Abcam), anti-β-Klotho (1:1000, Abcam), anti-β-actin (1:1000, Cell Signaling Technology), and anti-GAPDH (1:1000, Cell Signaling Technology). Subsequently, membranes were washed three times with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Protein signals were detected using a Tanon 5200 Chemiluminescence Imaging system (Tanon-5200) and bands were quantified using ImageJ software.
4
Western Blot Analysis of Islet Proteins
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Islet and cell proteins were extracted with CytoBuster Protein Extraction Reagent (Novagen, Darmstadt, Germany). Extracted proteins were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Bio-rad, Heidemannstraße, Germany), which were blocked with 5% milk and then probed overnight with anti-FGF21 (Abcam, Cambridge, UK), anti-LC3 (Novus, St. Charles, MO, USA), anti-phospho-AMPKα, anti-AMPKα, anti-phospho-ERK1/2, anti-ERK1/2, anti-phosphomTOR, anti-mTOR (Cell Signaling Technology, Boston, MA, USA), or anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibodies at room temperature. Horseradish peroxide-conjugated secondary antibodies were incubated at RT with the membranes for 2 h after washing with Phosphate buffered saline with Tween-20. Labeled protein bands were visualized on autoradiography films (Fuji Film, Tokyo, Japan) following application of ECL detection reagent (GE Healthcare, Chicago, IL, USA). The protein bands were quantitated in ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to β-actin. The primary and secondary antibodies used are listed in Table 2 .
5
Histological Evaluation of Kidney Tissue Damage
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Prior to histological analysis, kidney tissues were fixed in 4% paraformaldehyde for over 24 h, embedded in paraffin, cut into 5 μm sections, stained with hematoxylin and eosin (H&E), sealed with neutral resin, and observed under an optical microscope. Tissue damage and necrosis were assessed by microscopy (×400) in 10–20 randomly selected fields of view. Tissue damage was scored by the percentages of renal tubular injury, cell debris, brush border loss, and cast formation (0, no damage; 1, less than 25% damage; 2, 25–50% damage; 3, 50–75% damage; 4, more than 75% damage).
For immunohistochemistry analysis, 5 μm kidney tissue sections were incubated with anti-FGF21 (Abcam; Cambridge, United Kingdom) and anti-SIRT1 (Abcam; Cambridge, United Kingdom), followed by the secondary antibody (ZSGB-Bio, Beijing, China) and DAB. For each mouse, 10–20 randomly selected fields of view were scanned under an upright microscope. The percentages of positive staining regions of FGF21 or SIRT1 were quantified using Image-Pro 6.0.
For immunohistochemistry analysis, 5 μm kidney tissue sections were incubated with anti-FGF21 (Abcam; Cambridge, United Kingdom) and anti-SIRT1 (Abcam; Cambridge, United Kingdom), followed by the secondary antibody (ZSGB-Bio, Beijing, China) and DAB. For each mouse, 10–20 randomly selected fields of view were scanned under an upright microscope. The percentages of positive staining regions of FGF21 or SIRT1 were quantified using Image-Pro 6.0.
6
Immunoblot Analysis of Mouse Liver Proteins
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Mouse liver tissue or cultured cells were lysed with RIPA buffer and subjected to immunoblot analysis as described previously [47 (link)]. The membranes were probed with anti-ERRγ (Cell Signaling Technology, Danvers, MA, USA; diluted 1:1000), anti-β-actin (AbFrontier, Seoul, Korea; diluted 1:5000) [24 (link)] and anti-FGF21 (Abcam, Cambridge, UK; diluted 1:1000) [48 (link)] antibodies.
7
Comprehensive Protein Profiling Protocol
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The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874). All other antibodies were from Cell Signalling.
8
Western Blot Analysis of Hippocampal Proteins
9
Immunohistochemical Analysis of FGF21 and Vimentin
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Immunohistochemistry was performed as previously described (Geller et al., 2019 (link)). Mice were transcardially perfused with saline followed by 4% paraformaldehyde. Brains were collected and stored in 4% PFA overnight followed by 30% sucrose for 48 h. Coronal brain sections (50 μm) were collected using a cryostat (Leica). Free-floating sections were washed in 1X PBS and incubated in Citrate Buffer (Abcam) for 15 min at room temperature (RT), followed by 2 min at 60 C° and 90 C°. Slices were incubated in blocking buffer (5% donkey serum in PBST) for 1 h at RT and incubated in primary rabbit monoclonal anti-Fgf21 (1:100, Abcam) for three nights and chicken anti-Vimentin (1:2000, Millipore) overnight at 4 C°. Sections were subsequently washed and incubated in secondary antibody donkey anti-rabbit AF594 and donkey anti-chicken AF647 (Jackson Immunoresearch) or goat anti-rabbit Texas Red and goat anti-chicken AF488 for 2 h at RT. Slices were mounted on slides with Vectashield antifade mounting media with DAPI and imaged using an Olympus BX61 Light Microscope or an Olympus FV3000 confocal laser scanning microscope. Staining for cFos was performed as previously using a cFos antibody (1:1000, Cell Signaling) (Jensen-Cody et al., 2020 (link)).
10
Immunohistochemical Analysis of FGF21 and Vimentin
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