Protein expression was analyzed by Western blot analysis as described previously.12 (link) The primary antibodies used were as follows: anti-fibronectin (F3648, Sigma-Aldrich), anti-desmin (PB0095; Boster, Wuhan, China), anti-vimentin (SAB1305447, Sigma-Aldrich), anti-MMP-9 (AB805; Millipore), anti-podocin (sc-22298; Santa Cruz Biotechnology, Santa Cruz, CA), anti-podocalyxin (AF1556; R&D Systems), anti-ZO-1(QF215185; Life technologies), anti-nephrin(ab58968; Abcam), anti-Klotho (AF1819; R&D Systems), anti-p47phox (07-500; Millipore, Billerica, MA), anti-Nox2 (BA2811; Boster), anti-active β-catenin (05-665; Millipore), anti-Snail1 (ab17732; Abcam), anti-Wnt1 (ab15251; Abcam), anti-Wnt7a (sc-26361; Santa Cruz Biotechnology), anti-Flag(F3165; Sigma-Aldrich), anti-p65(4764S; Cell Signaling Technology), anti-p-p65(3031S; Cell Signaling Technology), anti-α-Tubulin (RM2007; Ray Antibody, Beijing, China) and anti-β-actin (MAB1501; Millipore, Billerica, MA).
Anti nephrin
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-nephrin is a laboratory research tool used to detect and quantify the nephrin protein. Nephrin is a key structural component of the glomerular slit diaphragm in the kidney. This product can be used in various techniques, including immunohistochemistry and Western blotting, to study the expression and localization of nephrin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti podocin, by Abcam (10 mentions)
Anti β actin, by Abcam (4 mentions)
Anti fibronectin, by Merck Group (2 mentions)
Anti podocin, by Santa Cruz Biotechnology (2 mentions)
Anti podocalyxin, by R&D Systems (2 mentions)
Anti p62, by Abcam (2 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Anti β catenin, by Abcam (2 mentions)
Ab58968, by Abcam (2 mentions)
Anti caspase 3, by Abcam (2 mentions)
Anti cyclin d1, by Cell Signaling Technology (2 mentions)
Anti fibronectin, by Abcam (2 mentions)
Anti p p38, by Cell Signaling Technology (2 mentions)
Anti p38, by Cell Signaling Technology (2 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (2 mentions)
Picric acid, by Solarbio (2 mentions)
Mayer s h e, by Nanjing Jiancheng (2 mentions)
Anti 53bp1, by Novus Biologicals (2 mentions)
Tunel kit, by Beyotime (2 mentions)
27 protocols using anti nephrin
1
Western Blot Analysis of Protein Markers
2
Kidney Tissue Protein Analysis
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The cortical part of the kidney tissue was taken and added with histone lysate for full grinding; then, the supernatant was taken, and the protein concentration was determined with BCA protein detection kit (Beyotime Institute of Biotechnology, Beijing). Then, 50 ng supernatant was added into 2 × SDS sample buffer to prepare the sample. After fully mixing, the sample was bathed in water at 100°C for 10 min. Cool to room temperature, set aside at 4°C, or store at -20°C. After sample loading, electrophoresis, membrane transfer, and sealing, specific anti-PPARγ (1 : 1000), anti-podocin (1 : 5000), anti-collagen-IV (1 : 500), anti-fibronectin (1 : 800) (Proteintech company), anti-nephrin (1 : 300) (Abcam), anti-β-actin (1 : 6000), (Bioworld Technology, Nanjing) were added, sealed, 4°C gently shake overnight. TBST was added to wash the membrane for 5 min × 3 times. The specific secondary antibody (ZSGB-Bio company) was incubated for 1 h, and the membrane was washed again with TBST for 5 min × 3 times. Add ECL (Beyotime Institute of Biotechnology, Beijing) development and exposure. Scans were performed using Shanghai Qinxiang gel imaging system. The ImageJ software analyzed the absorbance value of each band and detected the relative expression of each target protein, three times for each group.
3
Autophagy Regulation in Podocytes
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Rapamycin (RP) and chloroquine (CQ) were purchased from Sigma-Aldrich; Merck KGaA. Antibodies against LC3, mammalian target of rapamycin (mTOR) and p-mTOR were acquired from Cell Signaling Technology, Inc. Anti-Cx43, anti-podocin, anti-nephrin and anti-p62 antibodies were obtained from Abcam. Anti-GAPDH was purchased from CWBio.
4
Podocyte Autophagy Regulation Mechanisms
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Aldo, rapamycin (RP), chloroquine (CQ), 3-methyladenine (3-MA), tunicamycin (Tun), tauroursodeoxycholic acid (TAUDC), and anti-β-actin antibody were purchased from Sigma (St Louis, MO). Antibodies against LC3, Akt, p-Akt, mTOR, p- mTOR, S6K1, p-S6K1, 4EBP1, p-4EBP1, GRP78, GRP94, CHOP, FOXO1, p-FOXO1, Rab5, and Rab7 were purchased from Cell Signaling Technology (Beverly, MA). Anti-Podocin, anti-Nephrin, and anti-p62 antibodies were obtained from Abcam (Cambridge, MA). P300, Ac-FOXO1, and nestin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
5
Diabetic Kidney Disease Biomarker Analysis
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Serum creatinine (SCr) (lot#: C011-1-1), blood urea nitrogen (BUN) (#C013-1-1), serum uric acid (SUA) (#C012-1-1), triglyceride (TG) (#A110-1-1), total cholesterol (TC) (#A111-1-1), and urine protein (#C035-2-1) assay kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-α-smooth muscle actin (αSMA) antibody (#bs-0189R) was purchased from Beijing Biosynthesis Biotechnology Co.,Ltd. (Beijing, China). Anti-Rac1(#ab33186), anti-GTP-Rac1 (#ab33186), anti-p-PAK1 (#ab75599), anti-p38MAPK (#ab195049), anti-p-p38MAPK (#ab47363), anti-β-catenin (#ab32572), anti-Podocin (#ab50339), and anti-nephrin (#ab216341) antibodies were from Abcam (Cambridge, United Kingdom). Anti-β-actin (#66009-1-Ig), anti-fibroblast-specific protein-1 (FSP-1) (#16105-1-AP), anti-PAK1 (#21401-1-AP), anti-snail (#13099-1-AP), HRP goat anti-mouse IgG (#SA00001-1), and HRP goat anti-rabbit IgG (#SA00001-2) antibodies were from Proteintech (Chicago, United States). Trizol (#15596026) was from Thermo Scientific (MA, United States). A Reverse Transcription kit (#CW2569) was purchased from CWBio Co., Ltd. (Beijing, China). STZ (#WXBC8740V) was from Sigma-Aldrich Co. Ltd. (MO, United States). Metformin (#A181224) from Zhejian Yatai Pharmaceutical Co., Ltd. (Shaoxing, China).
6
Immunofluorescent Staining of Mouse Kidneys
7
Immunohistochemical Analysis of Autophagy and Podocyte Markers
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The kidneys were immersed in 4% paraformaldehyde and embedded in paraffin. Kidney cross-sections were dewaxed heated for antigen retrieval, incubated with 3% H2O2 to block endogenous peroxidase activity and blocked with 1% BSA. Then, the sections were incubated at 4°C overnight with the following primary antibodies: anti-LC3 (B) (Cell Signaling Technology, 1:50), anti-P62/SQSTM1 (Abcam, 1:100), anti-nephrin (Abcam, 1:100), and anti-podocin (Abcam, 1:100). The sections were incubated with the relevant secondary antibodies, and the positive staining was visualized by staining with diaminobenzidine (DAB) and counterstaining with hematoxylin and examined under an electron microscope at a magnification of 400×. All images were quantified with Image-Pro Plus 6.0 software.
8
Mitochondrial Proteome Analysis in Kidney
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The proteins of renal cortex tissue were lysed with Protein Extraction Kit (P0033, Beyotime, Jiangsu, China), the proteins of mitochondria and cytoplasmic were extracted with Tissue Mitochondria Isolation Kit (C3606, Beyotime, Jiangsu, China) and quantified by BCA protein assay kit (P1511, Applygen, Beijing, China). Total tissues lysates were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Afterward, the membranes were blocked with 5% BSA for 1 h at room temperature and followed by incubation with the primary antibodies anti-Parkin (SC-32282, Santa, Dallas, USA), anti-PINK1 (DF7742, Affinity, Ohio, USA), anti-LC3 (2775, CST, Boston, USA), SQSTM1/p62 antibody (5114, CST, Boston, USA), anti-Caspase-3 (9662S, CST, Boston, USA), anti-Cyt c (11940S, CST, Boston, USA), and anti-Nephrin (ab136894, Abcam, Cambridge, UK) overnight at 4 ℃. After washing, the corresponding HRPconjugated secondary antibodies were used. Finally, we used an ECL Kit (P1010, Applygen, Beijing, China) and a multi-functional imaging system to detect positive binding. Image-Pro Plus 6.0 software was used to quantify all protein bands.
9
Mitochondrial Dynamics in Kidney Injury
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Adriamycin (ADR, catalog number D1515) and dihydroethidium (DHE, catalog number D7008) were obtained from Sigma-Aldrich (St. Louis, MO). Hyperoside (HPS) was purchased Zelang Biological Technology Company (Nanjing, China). Anti-nephrin (catalog number ab58968), anti-podocin (catalog number ab50339), anti-PGC-1α (catalog number ab54481), phosphor Anti-Drp1 (S637) (catalog number ab193216) and anti-VDAC (voltage-dependent anion channel, catalog number ab34726) antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-Drp1 (catalog number sc-32898) and anti-Mfn-1 (catalog number sc-166644) antibodies were purchased from Santa Cruz (Santa Cruz, CA). anti-GAPDH was purchased from Sanying biotechnology (Wuhan, China, catalog number 10494-1-AP). All secondary antibodies for immunoblot analysis were from Zhongshan Golden Bridge Biotechnology (Beijing, China, catalog number ZB-2301). MitoSOX (catalog number M36008) and 2’,7’-dichlorofluorescein diacetate (DCFDA, catalog number C6827) were from Invitrogen (Carlsbad, CA, USA).
10
Quantitative Western Blot Analysis of Kidney Proteins
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Protein expression was analyzed by Western blot analysis of whole kidney lysates or whole cell lystaes as described previously (Zhou et al., 2014 (link)). The primary antibodies used were as follows: anti-podocalyxin (AF1556; R&D Systems), anti-ZO-1 (402200; Invitrogen), anti-nephrin (ab58968; Abcam), anti-WT1 (sc-393498; Santa Cruz Biotechnology), anti-fibronectin (F3648; Sigma), anti-α-SMA (ab5694; Abcam), anti-collogen Ⅰ (BA0325, Boster Biotechnology), anti-α-tubulin (RM2007; Ray Antibody Biotech) and anti-GAPDH (RM2002; Ray Antibody Biotech). Relative protein levels of Western blots were quantified with densitometries, analyzed by ImageJ software and reported after normalizing to the loading controls. Relative protein levels over the control group (setting as 1.0) were reported.
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