An ABC system (Dako, Glostrup, Denmark) was performed in paraffin-embedded human tissue as previously described.25 (link),27 (link),34 (link) Primary antibodies used include: CD34 (Covance, Princeton, NJ, USA), HIF-1α (ABCAM, Cambridge, MA, USA), ANGPTL-4 (ABCAM), and VEGF (Santa Cruz Biotechnology) after dilution in Tris-buffered saline (TBS) with 1% bovine serum albumen (BSA). All immunohistochemical reagents, including antibodies, were identical for all specimens.
Angptl4
Manufactured by Abcam
Sourced in United States, China
ANGPTL4 is a protein that plays a role in regulating lipid and glucose metabolism. It is involved in the inhibition of lipoprotein lipase activity. ANGPTL4 is expressed in various tissues, including the liver, adipose tissue, and skeletal muscle.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Abc system, by Agilent Technologies (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Hif 1α, by Abcam (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Methanol, by Merck Group (1 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
Aperio scanscope xt system, by Leica (1 mentions)
Lipopolysaccharides (lps), by Cusabio (1 mentions)
Cba mouse inflammation kit, by BD (1 mentions)
Lifespan, by LifeSpan BioSciences (1 mentions)
Blood urea nitrogen, by BioAssay Systems (1 mentions)
Glucometer, by Bayer (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Cfx96 system, by Bio-Rad (1 mentions)
8 protocols using angptl4
1
Immunohistochemical Analysis of Human Tissue
2
Molecular Mechanisms of Drug Interactions
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Methanol, crystal violet, and chloroquine (CQ) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal antibodies against cyclin-dependent kinase 4 (CDK4), poly(ADP-ribose) polymerase (PARP), Bcl-2, Bid, Thr(P)172-adenosine monophosphate-activated protein kinase (AMPK), AMPK, Ser(P)2448-mammalian target of rapamycin (mTOR), mTOR, Ser(P)79-acetyl-CoA carboxylase (ACC), ACC, fatty acid synthase (FAS), microtubule-associated light chain 3 (LC3), and p62 were obtained from Cell Signaling (Beverly, MA, USA); p21 and cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Ser(P)872-hydroxymethylglutaryl-CoA reductase (HMGCR), and HMGCR were from Millipore (Billerica, MA, USA); cyclin D1 was from Santa Cruz Biotechnology; and ANGPTL4 was from Abcam (Cambridge, MA, USA). Mouse monoclonal antibodies against beta-actin and caspase-3 were, respectively, purchased from MP Biomedicals (Irvine, CA, USA) and Imgenex (San Diego, CA, USA).
3
Immunohistochemical Detection of ANGPTL4 and CD34
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Immunohistochemical detection of ANGPTL4 (1:400 dilution; Abcam, Cambridge, MA) and CD34 (1:500 dilution; Covance, Princeton, NJ) was performed in paraffin-embedded tissue sections using premixed biotinylated anti-rabbit, anti-mouse and anti-goat immunoglobulins in phosphate buffered saline (PBS) from an ABC system (Dako, Santa Clara, CA) performed according to the manufacturer’s protocols as previously described[16 (link), 17 (link)]. All immunohistochemical reagents, including antibodies, were identical for all specimens. Images were captured by scanning slides using the Aperio ScanScope program on Aperio Scanscope XT® System (Leica Biosystems, Wetzlar, Germany).
4
Biochemical Analysis of Serum and Fecal Lipids
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Serum samples were tested to detect multiple biochemical parameters using commercial enzyme-linked immunosorbent assay kits, including CHOL (Abcam, Cambridge, UK), TG (Abcam), LPS (CUSABIO, Wuhan, China), HDL-C and LDL/VLDL-C (Abcam), and ANGPTL4 (Abcam).
Fecal lipid was determined in accordance with previous studies.39 In brief, fecal samples were collected from all groups when the mice were killed. After being dried at 60°C to a constant weight, samples were ground to a fine powder and then extracted 3 times with ethanol 95 % at 60°C. After filtration and evacuation, fecal TG and CHOL were detected by using commercial kits (Abcam).
Fecal lipid was determined in accordance with previous studies.39 In brief, fecal samples were collected from all groups when the mice were killed. After being dried at 60°C to a constant weight, samples were ground to a fine powder and then extracted 3 times with ethanol 95 % at 60°C. After filtration and evacuation, fecal TG and CHOL were detected by using commercial kits (Abcam).
5
Multi-Analyte Metabolic Profiling
6
Quantitative Gene and Protein Expression Analysis
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Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific). The purity of RNA was determined by NanoDrop ND‐1000 spectrophotometer (Thermo Scientific) qPCR was conducted using the appropriate primers and a Bio‐Rad CFX96 system with SYBR green to determine the mRNA expression levels of genes in cells. GAPDH was used as endogenous controls and further analysed by the 2∆DDCT method. All the primers used for qPCR are listed in the supplement data.
Total proteins were isolated from A549 and H1299 cells and dissolved in Ripa buffer which containing protease inhibitor (sigma, USA). The total protein concentration was determined by BCA analysis kit (Rockford Pierce, Illinois, USA). Total protein samples (30 mg) were analysed by 10% SDS‐PAGE gel and transferred to PVDF membrane. After the blocking procedure, the cell membrane was incubated overnight with primary antibody and secondary antibodies. Then, visualized in Imager (Bio‐Rad) using ECL system (Thermo Fisher Scientific, 34095). The following antibodies were used: ANGPTL4 (1:5000, abcam, MA, USA), GLS (1:1000, abcam, MA, USA), CPT1 (1:1000, abcam, MA, USA) and GAPDH (1:5000, abcam, MA, USA).
Total proteins were isolated from A549 and H1299 cells and dissolved in Ripa buffer which containing protease inhibitor (sigma, USA). The total protein concentration was determined by BCA analysis kit (Rockford Pierce, Illinois, USA). Total protein samples (30 mg) were analysed by 10% SDS‐PAGE gel and transferred to PVDF membrane. After the blocking procedure, the cell membrane was incubated overnight with primary antibody and secondary antibodies. Then, visualized in Imager (Bio‐Rad) using ECL system (Thermo Fisher Scientific, 34095). The following antibodies were used: ANGPTL4 (1:5000, abcam, MA, USA), GLS (1:1000, abcam, MA, USA), CPT1 (1:1000, abcam, MA, USA) and GAPDH (1:5000, abcam, MA, USA).
7
Immunofluorescence Analysis of ANGPTL4 in NP Cells
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Slides were placed in a six-well plate following disinfection with 75% ethanol to culture NP cells at a density of 3.5×105 per well. The NP cells were then treated with TNF-α (10ng/mL) or HGHF for 24 h after attachment and proliferation. After fixation with paraformaldehyde (4%) for 30 min, cell membranes were permeabilized with 0.25% Triton X-100 (Beyotime Biotechnology, China) for 10 min, blocked with 5% BSA for 2 h at room temperature, NP cells then incubated with the primary antibody ANGPTL4 (1:250, Abcam) overnight at 4°C. Cells were washed three times with PBS and then incubated with an Alexa Fluor 594 conjugate secondary antibody (anti-rabbit, 1:500; Cell Signaling Technology) for 1 h. Then, the cells were incubated with DAPI for 20 min. Eventually, fluorescence images were determined with a fluorescence microscope (Olympus, Inc., Tokyo, Japan). Semi-quantitative analysis of integrated optical density (IOD) was performed using Image-Pro Plus (version 6.0; Media Cybernetics, Silver Spring, MD, USA).
8
Quantification of Podocyte Proteins
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Cultured mouse podocytes and isolated renal glomerulus were lysed with RIPA buffer to gain total proteins. For cell culture supernatants, 10 ml of culture medium was concentrated with 50 ml Amicon® Ultra Centrifugal Filters (Millipore, MA, USA) to about 200 μl for further analysis. The protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Temecula, CA). PVDF membranes were blocked with 5% non-fat milk for 2 h and incubated with different primary antibodies. The primary antibodies were used as follows: Angptl4(1:1000, Abcam); synaptopodin (1:1000, Santa Cruz); CaN (1:500, Santa Cruz); GAPDH(1:5000, Bioke); β-actin (1:5000, Bioke); TLR4 (1:500, Santa Cruz); CD14 (1:1000, Cell Signaling); MyD88(1:1000, Cell Signaling); p-p65 (1:1000, Cell Signaling); p65 (1:1000, Cell Signaling); NFATc1 (1:500, Santa Cruz); HIF-1a (1:1000, Cell Signaling). After incubation with HRP-conjugated secondary antibodies for 2 h, membranes were visualized with an enhanced chemiluminescence detection system ChemiDoc MP (Bio-RAD, California, USA).
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