Isolated EV were lysed in mRIPA buffer (50 mM Tris pH 7.4, 1% NP‐40, 0.25% deoxycholate, 150 mmol/L NaCl, and protease inhibitors). 10 µg (for EV samples) or 30 µg (for cell samples) of protein lysate was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to BA85 nitrocellulose membrane (PROTRAN; Whatman). Proteins were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The following primary antibodies were used: CD144 (Santa Cruz Biotechnology), α‐Tubulin, β‐actin, α‐smooth muscle actin (α‐SMA), calponin, myosin heavy chain (MHC) (Sigma‐Aldrich), smooth muscle protein 22‐α (SM22α) (Abcam), Myocardin (R&D Systems), proliferating cell nuclear antigen (PCNA) (Proteintech Group), CD31, protein disulfide isomerase (PDI), endoplasmic reticulum (ER) stress protein 72 (ERp72), receptor‐binding cancer antigen expressed on SiSo cells (RCAS1), cytochrome c oxidase (COX) IV (Cell Signaling Technology, Danvers, MA). Detailed information about antibodies is in Table S1 .
Proliferating cell nuclear antigen (pcna)
Manufactured by Proteintech
Sourced in United States, China, United Kingdom, Germany
PCNA is a protein that plays a key role in DNA replication and repair processes. It acts as a DNA clamp, encircling the DNA and providing a platform for various enzymes involved in these cellular functions.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (29 mentions)
β actin, by Proteintech (15 mentions)
Bcl 2, by Proteintech (14 mentions)
E cadherin, by Proteintech (11 mentions)
Cyclin d1, by Proteintech (11 mentions)
Bcl2 associated x (bax), by Proteintech (10 mentions)
N cadherin, by Proteintech (10 mentions)
Ripa buffer, by Beyotime (9 mentions)
Vimentin, by Proteintech (9 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
Pvdf membrane, by Merck Group (7 mentions)
E cadherin, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Cyclin e1, by Proteintech (4 mentions)
C jun, by Cell Signaling Technology (4 mentions)
C myc, by Proteintech (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
P jnk, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
118 protocols using proliferating cell nuclear antigen (pcna)
1
Protein Expression Analysis of Extracellular Vesicles
2
Protein Extraction and Immunoblotting
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Total protein was extracted using a commercial lysis buffer (Thermo Scientific, MA, USA). Primary antibodies targeting phospho-c-Jun N-terminal kinase (p-JNK; Cell Signaling Technology, MA, USA), JNK (Cell Signaling Technology, MA, USA), phospho-extracellular signal-regulated kinase (p-ERK; Cell Signaling Technology, MA, USA), ERK (Cell Signaling Technology, MA, USA), p-p38 (Cell Signaling Technology, MA, USA), p38 (Cell Signaling Technology, MA, USA), tumour necrosis factor alpha (TNF-α; Cell Signaling Technology, MA, USA), cytochrome P450 2E1 (CYP2E1; Proteintech, Wuhan, China), proliferating cell nuclear antigen (PCNA; Proteintech, Wuhan, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, MA, USA) and actin (Cell Signaling Technology, MA, USA) were used.
3
Western Blot Analysis of Cellular Proteins
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Total protein, extracted from tissues or cells, was lysed in RIPA lysis buffer (Radio Immunoprecipitation Assay), which was composed of 50 mM NaCl, 0.1% Tween-20, 150 mM Tris-HCl, 1% NP-40 and a freshly added protease inhibitor cocktail (Sigma, USA). Before gel electrophoresis, the total protein concentration was determined with a Bio-Rad protein assay (BioRad, Hercules, CA, USA). An equal amount of protein was separated by 10% SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis) and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon, Millipore). After blocking for 2 h in 5% dried skim milk in Tris Buffer Solution Tween (TBST) at room temperature, the membranes were then incubated overnight with primary antibodies against NDC1 (1:1,000, Thermo Fisher Scientific, USA), Proliferating Cell Nuclear Antigen (PCNA) (1:1,000, Proteintech, Wuhan, China), E-cadherin (1:1,000, Cell Signaling, USA), vimentin (1:1,000, Cell Signaling), and GAPDH (1:5,000, Proteintech, Wuhan, China). After incubating with an anti-rabbit/mouse immunoglobulin G (IgG) (Thermo Fisher Scientific) and a horseradish peroxidase-conjugated secondary antibody, the protein was visualized by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific).
4
Glomerular Mesangial Cell Proliferation Assessment
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Kidney tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin–eosin (H&E) and periodic acid‐Schiff (PAS) staining was performed to observe the proliferation of glomerular mesangial cells. According to the instructions of the immunohistochemistry kit (ZSGB‐Bio, China), the expressions of cyclin D1 (1:100, Abcam, USA), PCNA, P62 (1:100, Proteintech, China), and LC3 II (1:100, Servicebio, China) were detected. Quantitative analysis was performed using ImageJ software.
5
Western Blot Analysis of Adipogenic Markers
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Cells were lysed on ice in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Beyotime, Haimen, China). Total and nuclear proteins were extracted according to the manufacturer’s instructions (Beyotime, Haimen, China). Protein samples (50 μg) were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked in TBST containing 5% fat-free milk at room temperature for 1 h. After washing with TBST, the membranes were probed with primary antibodies against C/EBPα, FABP4, PPARγ, RhoA, ROCK1, ROCK2, ERK1/2, β-actin, PCNA (all Proteintech, Wuhan, China), and p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA). The membranes were washed in TBST and incubated with secondary antibody (Proteintech, Wuhan, China). The signals were developed by ECL reagents (Beyotime, Haimen, China).
6
Protein Expression Analysis in Cell Samples
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Samples were homogenized using RIPA buffer (Beyotime, Shanghai, China) with Protease and Phosphatase Inhibitor (Thermo Fisher) or Nuclear-Cytosol Extraction Kit (Fdbio science, Hangzhou, China). Proteins were separated in SDS-PAGE and then transferred to the PVDF membranes (Merck Millipore). After blocking with 5% bovine serum albumin, the membranes were incubated with antibodies raised against Estrogen Receptor alpha (1:1000, ab32063, Abcam, USA), PCNA (Proteintech, USA), NLRP3 (1:1000, ab263899, Abcam), cleaved caspase-1 (1:1000, 89332, Cell Signaling Technology), pro-caspase-1 (1:1000, ab179515, Abcam), IL-1β (1:1000, 12242, Cell Signaling Technology), ASC (1:1000, 67824, Cell Signaling Technology), and GAPDH (1:1000, Mab5465, MultiSciences, Hangzhou, China). After incubation with HRP-conjugated secondary antibody against rabbit IgG (1:10000, ab97051, Abcam) or against mouse IgG (1:10000, ab97023, Abcam), the membranes were detected using the ECL detection kit (Biological Industries) and imaged on the ImageQuant LAS 4000 Mini Biomolecular Imager (GE Healthcare Life Sciences, USA). The detected protein expression was quantified on band volume by Image J and normalized over GAPDH mean band intensity.
7
Western Blot Analysis of Cell Cycle Regulators
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One hundred microgram protein lysate per lane was used for Western blot analysis. Antibodies used in this study: RB1 (CusaBio PA003948), RB-P (Cell Signaling 9307), E2F1 (Santa Cruz SC-193), CDK2 (CusaBio PA001533), CDK4 (Santa Cruz SC-23896), CDK6 (Santa Cruz SC-53638), CCND1 (Santa Cruz SC-8396), CCNE2 (Proteintech 11,935–1-AP), CDKN2A (Prosci 4211), CDKN1A (Santa Cruz SC-6246), PCNA (Proteintech 10,205–2-AP) and GAPDH (Santa Cruz FL-335).
8
PDGF-Mediated Signaling Pathway Investigation
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Platelet-derived growth factor (PDGF) was purchased from Proteintech (Wuhan, China); U0126 was provided by Selleck Chemicals (Shanghai, China); fetal bovine serum (FBS), RPMI-1640, and penicillin-streptomycin solution were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA); ovalbumin (OVA) was obtained from Sigma-Aldrich (Saint Louis, MO, United States); and dexamethasone (DEX) was purchased from Xianju Pharmaceuticals (Zhejiang, China). p-ERK1/2, ERK1/2, and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA); p-MEK1/2, MEK1/2, and β-actin antibodies were obtained from Affinity (Changzhou, China); PCNA, α-SMA, and GAPDH antibodies were purchased from Proteintech (Wuhan, China); and RIPA lysis buffer and BCA protein assay kit were obtained from Beyotime (Shanghai, China).
9
Western Blot Analysis of Renal Cell Proteins
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A total of 50 μg of total renal cell lysate was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore; Billerica, MA, USA). Western blotting was performed as described in our previous study [19 (link)]. The following primary antibodies were used: PCNA (diluted 1:500, Proteintech, USA, # 60097-1-Ig), Bcl-2 (diluted 1:1000, Santa Cruz, #sc-7382), and Bax (diluted 1:1000, Santa Cruz, #sc-7480). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (diluted 1:3000; Amyjet, #AS09-602 and AS10-1427) were used to detect proteins using an ECL Assay Kit (Bipec Biopharma). β-Tubulin (diluted 1:1000; Proteintech, USA, # 10094-1-AP) or β-actin (diluted 1:1000; Proteintech, USA, #20536-1-AP) was used as an internal control. Band intensity was quantified using the ImageJ software (1.44 P).
10
Molecular Profiling of EMT Markers
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Total protein was extracted by lysing cells in RIPA buffer containing protease inhibitors. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk in TBST, membranes were incubated with the primary antibody. Antibodies against ZEB1, cleaved caspased-3, E-cadherin, N-cadherin, Vimentin, Snail and Slug purchased from Cell Signaling Technology in USA were diluted at 1:1000. Bcl-2, Bax, PCNA, UBQLN1, MMP2, MMP9 and β-actin purchased from Proteintech Group in USA were diluted at 1:1000 and Peroxidase-conjugated Affinipure goat anti-mouse IgG (H+L) or anti-rabbit IgG (H+L) were diluted at 1:4000 as the secondary antibody.
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