Protein expression was analyzed using Western blotting as previously described (Yu et al. 2019 (link); Gong, Yan, et al. 2019 (link)). For Western blot analysis, primary antibodies against IL-6 (mouse monoclonal antibody, ab9324) and nephrin (rabbit monoclonal antibody, ab216341) were purchased from Abcam (UK), Anti-TGF-β1 (mouse monoclonal antibody, sc-130348) was purchased from Santa Cruz (USA), anti-podocin (rabbit polyclonal antibody, TA351459) was purchased from Origene (USA), anti-caspase-3 (rabbit polyclonal antibody, 9662S) was purchased from CST (USA), and anti-ALB (rabbit polyclonal antibody, 16475-1-AP) was purchased from Proteintech (USA). Goat anti-mouse IgG-HRP (ZB2305) and goat anti-rabbit (ZB2301) IgG-HRP secondary antibodies were purchased from ZSGB-Bio (Beijing, China). The targeted proteins were visualized with the Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific Pierce), and the intensities of the visualized bands were analyzed using Quantity One software (Bio-Rad). β-Actin (mouse monoclonal antibody, TA-09, from ZSGB-Bio in Beijing, China) was used as an internal control. The data are expressed as the ratio to β-Actin.
Ab216341
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab216341 is a recombinant monoclonal antibody that can be used as a tool for research purposes. The product details and technical specifications are available on the Abcam website.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab50339, by Abcam (3 mentions)
Ab9324, by Abcam (2 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Ab32503, by Abcam (2 mentions)
Nephrin, by Abcam (2 mentions)
Zli 9018, by ZSGB-BIO (2 mentions)
Ab212951, by Abcam (2 mentions)
Ab181143, by Abcam (2 mentions)
Sc 130348, by Santa Cruz Biotechnology (1 mentions)
Anti alb, by Proteintech (1 mentions)
Goat anti rabbit zb2301 igg hrp secondary antibodies, by ZSGB-BIO (1 mentions)
Anti tgf β1, by Santa Cruz Biotechnology (1 mentions)
Ta 09, by ZSGB-BIO (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
Goat anti mouse igg hrp zb2305, by ZSGB-BIO (1 mentions)
Enhanced chemiluminescence working solution, by Merck Group (1 mentions)
Ab183612, by Abcam (1 mentions)
Ab219412, by Abcam (1 mentions)
25 protocols using ab216341
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Alzheimer's Biomarkers
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Rat cerebral tissues or cell lysates of the above groups were placed in ice-precooled RIPA lysis buffer (Beyotime) for 30 min, followed by centrifugation (12000 g, 10 min, 4° C). The supernatant was used for analysis. Protein concentration was determined using the BCA (Beyotime) method. Proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime) for separation, transferred to the nitrocellulose membranes (Whatman Inc., Piscataway, NJ, USA), and then blocked for 1 h with blocking solution (P0023B, Beyotime) at room temperature. Afterwards, the membranes underwent an overnight incubation with primary antibodies at 4° C, followed by membrane washing and then a 2-h incubation at room temperature with secondary antibody horseradish peroxidase-labeled immunoglobulin G (IgG) H&L (ab205718, 1:5000, ABCAm). The enhanced chemiluminescence working solution (EMD Millipore Corporation, Billerica, MA, USA) was used for development. ImageJ 1.48 (National Institutes of Health) was used for data analysis with β-actin as an internal reference. The primary antibodies (all from ABCAm) were neprilysin (NEP) (ab216341, 1:1000), insulin-degrading enzyme (IDE) (ab133561, 1:1000), BACE1 (ab183612, 1:1000), Wnt3a (ab219412, 1:1000), β-catenin (ab32572, 1:5000) and β-actin (ab8227, 1:1000).
3
Protein Expression Analysis in Tissue/Cells
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Sirt6, Nephrin, Desmin, CD86, CD206 proteins were extracted from the tissues or cells using RIPA lysis buffer (Beyotime, Shanghai, China). The concentrations of proteins were detected using the BCA protein kit (Beyotime, Shanghai, China). Aliquot protein was separated by 12% SDS-PAGE and resolved proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), which were blocked in 5% skim milk PBS with 0.1% Triton X-100 and incubated with primary antibodies as follows: anti-Nephrin antibody (1:1,000; ab216341), anti-Desmin antibody (1:1,000; ab15200), anti-Sirt6 antibody (1:2,000; ab191385), anti-CD86 antibody (1:5,000; ab53004 and ab112490), anti-CD206 antibody (1:1,000; ab64693), anti-Bcl-2 antibody (1:2,000; ab182858) and anti-Bax antibody (1:1,000; ab32503) (all from Abcam) overnight at 4°C. The membranes were then incubated with the appropriate HRP-conjugated secondary antibody (Proteintech Group, Inc./Thermo Fisher Scientific, SA00001-2, 1:5,000). Protein bands were detected with ECL (Thermo Fisher Scientific) and visualized using Quantity One software (Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Kidney Proteins
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Kidney tissue samples were thoroughly homogenized in RIPA lysis buffer (P0013B, Beyotime Biotech Co., Ltd., Shanghai, China) at low temperature. The protein concentrations in supernatants were measured using a BCA Protein Assay Kit (P0012S, Beyotime, Shanghai, and China). Protein aliquots (20 μg) were separated by 8–15% SDS-PAGE, transferred onto polyvinylidene difluoride membrane (Merck Millipore, United States), and blocked in 5% skim milk in Tris-buffered saline with Tween 20 (TBST). Then, blots were incubated overnight at 4°C in primary antibody solution containing anti -nephrin (ab216341), -AURKB (ab2254, Abcam, Cambridge, United Kigdom), podocin (20384-1-AP), -RacGAP1 (13739-1-AP), -RhoA (10749-1-AP), -Shcbp1 (12672-1-AP; 1:1,000) (all from Proteintech, Wuhan, China), or -GADPH (1:2,000, AF7021, Affinity, Jiangsu, China). Then, blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:1,000, 7074S, Cell Signaling Technology, MA, United States) for 2 h at 4°C. Protein bands were visualized using an ECL chemiluminescence reagent kit (WBKLS0100, Millipore, MA, United States). The average optical density of bands was quantified using ImageJ (National Institutes of Health, Bethesda, United States).
5
Immunofluorescence Analysis of Nephrin
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Following previous work [41 (link)], cell slides were prepared and fixed with 4% paraformaldehyde. Following rinsing with PBS, the slides were sealed with goat serum at room temperature for 30 min and then incubated with the anti-Nephrin (1:500, ab216341, Abcam) overnight at 4 °C. The slides were washed in PBS with 0.05% Tween-20 three times and next incubated with goat anti-rabbit secondary antibody Alexa Fluor® 594 IgG H&L (2 μg/mL, ab150080) in dark conditions. The nuclei were stained with 4′,6-diamidino-2-phenylindole and photographed under a fluorescence microscope.
6
Immunohistochemical Analysis of Nephrin in Renal Tissue
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The slices of kidney tissue were dewaxed and rehydrated, followed by antigen retrieval with Tris-EDTA (PH9.0) at a high pressure. Hydrogen peroxide (3%) was used to block the endogenous peroxidase for 15 min. After 30-min blocking with 10% goat serum at 37°C, the renal slices were incubated using following primary antibodies nephrin (1:250, ab216341, Abcam, UK) overnight at 4°C. Next, 1-hr secondary antibody incubation was performed. Then, the renal tissue sections were detected using Max VisionTM HRP kit (MXB Biotechnologies, Fujian, China) and stained with hematoxylin (Leagene, Beijing, China). The stainings were visualized by microscope (Bio-Rad, Laboratories, Hercules, CA, United States), and quantitation was conducted by Image J.
7
Kidney Protein Isolation and Analysis
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Total protein from the kidney tissues was isolated after being lysed by RIPA buffer (01408, Beyotime Biotechnology, China) containing PMSF, protease, and protein phosphatase inhibitor for 30 min on ice. All samples were centrifuged at 12000 g for 15 min (4°C). Protein concentration was determined using the BCA assay kit (P0012, Beyotime Biotechnology, China). An equal amount of protein (40 μg) was subjected to 8–10% SDS-PAGE gel and then transferred to PVDF membranes (162–0177, BIO-RAD, USA). The membranes were blocked with 5% BSA and incubated overnight with the following antibodies at 4°C: antinephrin (ab216341, Abcam, Cambridge, UK), anti-Wilms Tumor 1 (WT1) (ab89901, Abcam, Cambridge, UK), anti-Aquaporin 1 (ab168387, Abcam, Cambridge, UK), and anti-GAPDH (EM1101, Huaan Biotechnology, Hangzhou, China). After incubating with peroxidase-conjugated secondary antibodies (goat anti-rabbit-antibody, LI-COR Biosciences, USA), the protein expression bands were developed with chemiluminescence. The densitometry of the brands was then calculated using the Odyssey near-infrared dual-color laser imaging system (Odyssey Clx, LI-COR Biosciences, USA).
8
Immunofluorescence Analysis of Podocytes
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The podocytes were plated in a 24-well plate. When 40% confluent, the cells were fixed, infiltrated and blocked. Each slide was incubated with anti-nephrin (ab216341, 1:500, Abcam, USA) and anti-podocin (ab50339, 1:500, Abcam, USA) at 4 °C overnight. Then the slides were washed three times with PBS and incubated with Alexa Fluor 488 labeled goat anti-rabbit IgG (A0423, 1:100, Beyotime, Shanghai, China) for 45 min at room temperature. Finally, cells were observed under a confocal laser scanning microscope (Olympus, FV 1000, Center Valley, PA, USA).
9
Immunohistochemical Analysis of Kidney Tissue
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All kidney tissues were fixed overnight in formalin solution, dehydrated in ethanol, embedded in paraffin and then sectioned at 5 mm. The slides were blocked with 5% normal goat serum (Gibco, Thermo Fisher Scientific, Inc.) for 15 min at room temperature and incubated overnight at 4°C with primary antibodies, as follows: Anti-α-SMA (bs-0189R; 1:200; Bioss Biotechnology Co., Ltd., Beijing, China), anti-vimentin (ab137321; 1:100; Abcam, Cambridge, MA, USA), anti-nephrin (ab216341; 1:100; Abcam), and anti-podocalyxin-like 1 (PODXL1; ab197769; 1:100; Abcam). Subsequent to washing with PBS, the slides were incubated with the rabbit anti-rat horseradish peroxidase (HRP)-conjugated IgG secondary antibody (ab6734; 1:500; Abcam) at room temperature for 20 min. A DAB kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to detect immunohistochemical reactions. The slides were then examined under a phase contrast light microscope (Eclipse E600; Nikon Corp.) at ×200 magnification.
10
Quantifying Podocyte Protein Levels
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Cell and tissue lysates were prepared using RIPA buffer and equal protein amounts resolved on 10% SDS-PAGE gels before transfer to PVDF membranes. The membranes were then blocked and incubated with primary antibodies against nephrin (catalog no. Ab216341, Abcam, UK), beclin1 (catalog no. NB500-249 Novus, USA), podocin (catalog no. Ab181143 Abcam, UK), synaptopodin (catalog no. sc-515842 Santa Cruz, USA), desmin (catalog no. Ab8592 Abcam, UK), LC3 (catalog no. NB100-2220 Novus, USA) and Atg16L1 (catalog no. A1871; ABdonal) overnight. Protein bands levels were detected and analyzed using Image-Pro plus software with β-actin used as the internal reference.
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