At 24 h post-treatment with Huc-MSCs-exo, protein was extracted from cells using the triplePrep kit (cat. no. 28-9425-44; ReadyPrep; GE Healthcare Life Sciences). The protein levels were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. A total of 25 µg/lane protein was separated via SDS-PAGE on a 10% gel and transferred onto nitrocellulose membranes. The membranes were blocked using 5% skimmed milk at room temperature for 2 h and incubated overnight at 4°C with the following primary antibodies: Rabbit polyclonal anti-E-cadherin (1:1,500; cat. no. AF0131; Affinity Biosciences), anti-GAPDH (1:1,000; cat. no. AG019; Beyotime Institute of Biotechnology), anti-Vimentin and anti-N-cadherin (1:3,000 and 1:1,000, respectively; cat. nos. ab92547 and ab76057, respectively; both Abcam). After washing with 0.1 M PBS, the membranes were incubated with the secondary antibody (HRP-labeled goat anti-rabbit IgG; cat. no. A16104; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The blots were visualized using an electrochemiluminescence kit (Thermo Fisher Scientific, Inc.). The densities of the blots were quantified using the Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.).
Anti n cadherin
Manufactured by Beyotime
Sourced in China
Anti-N-cadherin is a lab equipment product that is used to detect the presence of N-cadherin, a cell adhesion protein, in biological samples. It functions as a specific antibody that binds to N-cadherin, allowing its identification and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Anti vimentin, by Beyotime (2 mentions)
Anti e cadherin, by Beyotime (2 mentions)
Ecl kit, by Beyotime (2 mentions)
Anti β actin, by Beyotime (2 mentions)
Electrochemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Affinity Biosciences (1 mentions)
Readyprep, by GE Healthcare (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Ab135246, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Anti mmp9, by Beyotime (1 mentions)
Af6285, by Beyotime (1 mentions)
Af1552, by Beyotime (1 mentions)
Anti bcl 2, by Beyotime (1 mentions)
Af5234, by Beyotime (1 mentions)
Anti mmp2, by Beyotime (1 mentions)
Anti gapdh, by Beyotime (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Anti β actin, by Abcam (1 mentions)
4 protocols using anti n cadherin
1
Protein Expression Analysis of Huc-MSCs-exo
2
Western Blot Analysis of Protein Expression
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Western blot analysis was performed to detect protein expression levels. Proteins were extracted from cell lysates using a cell lysis buffer (Beyotime, Shanghai, China) and separated by 10% SDS-PAGE followed by electrotransfer onto nitrocellulose membranes, blocking with skimmed milk at room temperature for 1 h. Membranes were incubated overnight at 4°C with primary antibodies, including anti-Nectin2 (ab135246; Abcam, Cambridge, USA), anti-GAPDH (AF1186; Beyotime), anti-β-actin (ab8226; Abcam), anti-MMP2 (AF1420; Beyotime), anti-MMP9 (AF5234; Beyotime), anti-cleaved caspase-3 (ab32042; Abcam), anti-bax (bs-0127R; Bioss, Beijing, China), anti-bcl2 (AF6285; Beyotime), anti-N-cadherin (AF0243; Beyotime), anti-E-cadherin (AF1552; Beyotime) and anti-ANXA2 (AF5115; Beyotime). Membranes were washed several times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (KC5G5; Aksomics, Shanghai, China) at room temperature for 1 h. Protein blots were visualized using an ECL kit (P0018FS; Beyotime), and protein levels are expressed as the ratio of band optical intensity relative to that of GAPDH. Densitometry analyses were performed using ImageJ software (version 1.52; NIH, Bethesda, USA).
3
Investigating EMT Markers in Cell Lines
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See below for all antibodies used: anti-KIFC1 (Ther-moFisher, cat. 20790-1-AP, Waltham, MA, USA), anti-Ecadherin (Beyotime, cat. AF6759, Shanghai, China), anti-N-cadherin (Beyotime, cat. AG1554, Shanghai, China), anti-Vimentin (Beyotime, cat. AF1975, Shanghai, China), anti-TGFβ (Beyotime, cat. AF0297, Shanghai, China), anti-SMAD2/3 (Beyotime, cat. AF8001, Shanghai, China), anti-phospho-Smad 2/3 (Beyotime, cat. AF5920, Shanghai, China), anti-SMAD4 (Beyotime, cat. AF1291, Shanghai, China), anti-β actin (Beyotime, cat. AF5003, Shanghai, China), Secondary antibody (Beyotime, cat. A0208, Shanghai, China).
4
Quantification of Podocyte Protein Levels
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The collected kidney tissues and podocytes from diverse groups were lysed with RIPA Buffer on ice for 20 min at 4°C to obtain the total protein. After determining the concentration of total protein, equivalent amounts of protein were separated on 10% SDS-PAGE gels and subsequently transferred onto PVDF membranes. Membranes were blocked with skimmed milk and then incubated with primary antibodies as followes:
anti-SIRT1 (#AF0282, 1:1000; Beyotime), anti-β-actin (#AF0003, 1:1000; Beyotime), anti-Desmin (#AF1414, 1:2000; Beyotime), anti-Nephrin (#PA5-106921, 1:2000;
Thermo Fisher Scientific, Inc.), anti-Synaptopodin (#PA5-21062, 1:2000; Thermo Fisher Scientific, Inc.), anti-P-cadherin (#13-2000Z, 1:1000; Thermo Fisher Scientific, Inc.), and anti-N-cadherin (#AF0243, 1:800; Beyotime). Then, the membranes were rinsed thrice before incubation with HRP-labeled Goat Anti-Rat IgG secondary antibody.
Finally, by using an ECL kit (Beyotime), the protein bands were visualized, of which intensities were measured by Image J 6.0 software..
anti-SIRT1 (#AF0282, 1:1000; Beyotime), anti-β-actin (#AF0003, 1:1000; Beyotime), anti-Desmin (#AF1414, 1:2000; Beyotime), anti-Nephrin (#PA5-106921, 1:2000;
Thermo Fisher Scientific, Inc.), anti-Synaptopodin (#PA5-21062, 1:2000; Thermo Fisher Scientific, Inc.), anti-P-cadherin (#13-2000Z, 1:1000; Thermo Fisher Scientific, Inc.), and anti-N-cadherin (#AF0243, 1:800; Beyotime). Then, the membranes were rinsed thrice before incubation with HRP-labeled Goat Anti-Rat IgG secondary antibody.
Finally, by using an ECL kit (Beyotime), the protein bands were visualized, of which intensities were measured by Image J 6.0 software..
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