We first utilized 5 ng/ ml TGF-β1 (RP00161, ABclonal, United States) to treat cells for 0, 24, 48, and 72 h and observed the alterations of cellular morphology and EMT-related markers at 72 h. Then, to check the roles of serum-containing MSJZD on cells, the cells were assigned to the blank group (cells were exposed to 10% control rat serum), TGF-β1 group (cells were subjected to 5 ng/ ml TGF-β1 and 10% control serum), low-dose group (cells were subjected to 5 ng/ ml TGF-β1 and low-dose serum-containing 10% MSJZD), medium-dose group (cells were stimulated with 5 ng/ ml TGF-β1 and medium-dose serum-containing 10% MSJZD), and high-dose group (cells were exposed to 5 ng/ ml TGF-β1 and high-dose serum-containing 10% MSJZD).
Tgf β1
Manufactured by ABclonal
Sourced in United States, China
TGF-β1 is a recombinant protein produced by ABclonal. It is a member of the transforming growth factor beta (TGF-β) family and plays a role in cellular processes such as cell growth, cell differentiation, and immune function.
Automatically generated - may contain errors
Lab products found in correlation
Smad2, by ABclonal (3 mentions)
Smad3, by ABclonal (3 mentions)
P smad3, by Abcam (3 mentions)
P smad2, by ABclonal (2 mentions)
P smad3, by ABclonal (2 mentions)
Gel pro analyzer, by Media Cybernetics (2 mentions)
α sma, by Proteintech (2 mentions)
Col1a2, by ABclonal (2 mentions)
α sma, by ABclonal (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Smad7, by ABclonal (1 mentions)
Igg hrp, by Beyotime (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Hy 12364, by MedChemExpress (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Proteinext mammalian nuclear and cytoplasmic protein extraction kit, by Transgene (1 mentions)
Hmgb1, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
12 protocols using tgf β1
1
Investigating TGF-β1-Induced EMT Modulation
2
Comprehensive Protein Analysis in Cell and Tissue Samples
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The specific procedures for cell and tissue protein extraction, quantification and western blot have been described in detail in our previous research [11 (link)]. The information of antibodies is as follows: IGF1 (1: 1000, Abclonal, #A11985, China), IGF1R (1: 1000, CST, #9750, USA), p-IGF1R(Tyr1135/1136) (1: 1000, CST, #3024, USA), COL-1 (1: 2000, Abcam, ab260043, UK), COL-3 (1: 1000, Abcam, ab184993, UK), α-SMA (1:1000, CST, #19425, USA), heme oxygenase-1 (HO-1, 1: 1000, CST, #5853, USA), N-cadherin (1: 1000, CST, #49398, USA), E-cadherin (1: 1000, CST, #49398, USA), TGFβ1 (1: 1000, abclonal, #A2124, China), p-NF-kB p65(Ser536) (1: 1000, CST, #3033, USA), NF-kB p65 (1: 1000, CST, #8242, USA), NLRP3 (1: 1000, CST, #15101, USA), Cleaved Caspase1(Asp297) (1: 1000, CST, #4199, USA), Caspase1 (1: 1000, CST, #2225, USA), p-Akt(Ser473) (1: 1000, CST, #4060, USA), Akt (1: 1000, CST, #4691, USA), p-GSK3β(Ser9) (1: 1000, CST, #9322, USA), GSK3β (1: 1000, CST, #9369, USA) and β-actin (1: 1000, CST, #4970, USA).
3
Protein Expression Analysis by Western Blot
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The concentration of the extracted protein was measured using a BCA protein assay kit (Beyotime, China) according to the manufacturer's protocol. After SDS-polyacrylamide gel electrophoresis, the separated protein was transferred onto polyvinylidene difluoride membranes (Millipore, USA) at 80 V for 1.5 h. The protein was then subjected to immune blot analysis with specific antibodies for ITGBL1 (Abclonal, China), Smad2 (Abclonal), p-Smad2 (Abclonal), Smad3 (Abclonal), p-Smad3 (Abclonal), Smad7 (Abclonal), JDP2 (Affinity, China), TGF-β1 (Abclonal), Ki67 (Abclonal), MMP2 (Proteintech, China), and β-actin (Santa Cruz, USA). All antibodies were diluted to 1:1000, except JDP2, which was diluted to 1:2000. In addition, the secondary antibody reagent in this study was IgG-HRP (Beyotime, China) with 1:5000 dilutions.
4
CPT1A Modulates TGF-β1-Induced EMT in HK-2 Cells
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HK-2 cells (TCH-C400) were obtained from Haixing Biosciences (Suzhou, China) and cultured in high glucose DMEM containing 10% FBS and 1% penicillin‒streptomycin. The cells were maintained at 37 °C in a 5% CO2 atmosphere. Briefly, HK2 cells were firstly cultured for 24 h to reach about 80% confluence. New DMEM without FBS were then used for HK-2 starvation overnight before being treated with TGF-β1 (10 ng/ml, ABclonal, China) to induce the EMT process, which mimics the process in kidney fibrosis. The cells in some experiments were pretreated with the Carnitine palmitoyltransferase 1 A (CPT1A) activator C75 (10 µM, HY-12,364, MedChemExpress, USA) or the CPT1A antagonist Etomoxir sodium salt (80 µM, HY-50,202 A, MedChemExpress, USA) for 2 h [28 (link)].
5
Quantitative Protein Analysis in Liver
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Total protein was extracted from liver tissues, and cells were lysed with RIPA buffer, PMSF (Beyotime, China) and Cocktail (Biotool, China). Nuclear extracts were isolated with the Protein Ext Mammalian Nuclear and Cytoplasmic Protein Extraction Kit (TransGen Biotech). The protein from each sample was selected by electrophoresis in 8–12% SDS-PAGE gels (Bio-Rad, United States). These strips were cultured overnight with the designated primary antibodies: HMGB1 (Cell Signaling Technology, United States), TLR4 (Proteintech, China), NF-κB p65 (Proteintech, China), α-SMA (Proteintech, China), Col-1 (Abcam, British), TGF-β1 (ABclonal, China), MMP9 (Wanleibio, China), TGF-β R1 (Wanleibio, China) and GAPDH (Proteintech, China). After incubation with corresponding secondary antibodies at 37°C, bands were exposed and developed after the addition of enhanced chemiluminescence-plus reagents (Advansta, United States). Western blot (WB) images were analyzed with a Gel-Pro Analyzer (Version 5.0; Media Cybernetics, Rockville, MD, United States).
6
Fibrosis Protein Expression Analysis
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LF tissues were ground using liquid nitrogen, and cells were washed using PBS. Suspended cells (in RIPA lysis buffer) were lysed on ice. An 8% separation gel and a 5% stacking gel were used for electrophoresis. Through the wet transfer method, separated proteins were transferred to nitrocellulose membranes. Membrane blocking was achieved using 5% milk for 1 h, after which they were incubated with primary antibodies against TGF-β1 (1:1000, ABclonal, China), COL1A2 (1:1000, ABclonal), a-SMA (1:1000, ABclonal), CLU (1:1000, ABclonal), ALK5 (1:1000, ABclonal), SMAD3 (1:2000, Abcam, UN), p-SMAD3 (1:2000, Abcam) and GAPDH (1:1000, ABclonal) at 4 °C overnight. Visualization was conducted using enhanced chemiluminescence (Affinity, USA), while a Quantity One system (Syngene, UN) was used for quantitative analysis of proteins.
7
Antibody Profiling of Signaling Pathways
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The following antibodies were purchased: CDH11 (Abcam, 4442), vimentin (Abcam, ab8978), collagen-III (Abcam, ab34710), α-SMA (Abcam, ab5694), JNK (Cell Signaling, #9252), pJNK1/2 (Cell Signaling, #9251), ERK1/2 (Abclonal, A10613), pERK1/2 (Abclonal, AP0472), P38 (Abclonal, A11340), pP38 (Abclonal, AP0297), Smad2/3 (Cell Signaling), p-Smad2 (Abcam), p-Smad3 (Abcam), Smad4 (Cell Signaling), TGF-β1 (Abclonal), TGF-βR1 (Abclonal), GAPDH (Beyotime, AG019) and goat anti-rabbit IgG H&L (Beyotime, A0423). Angiotensin-II (A9525) was purchased from Sigma-Aldrich and hIgG1 (110-HG) and hCDH11-Fc (1790-CA) were from R&D Systems. LY3200882 (TGF-β-Smad pathway inhibitor), U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) and SB203580 (P38-MAPK inhibitor) were purchased from Selleck Chemicals (USA).
8
Renal Cortex Protein Expression Analysis
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Changes in protein expression in renal cortex homogenates or urine samples were assessed using primary antibodies (nephrin, 1:1000, #ab58968, Abcam; β arrestin 1 1:1000, #A0998, Abclonal; phospho-β arrestin 1, 1:1000, # 2416, Cell Signaling; p44/42 MAPK (Erk1/2), 1:1000, #9102, Cell Signaling; phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), 1:1000, #9101 Cell Signaling; JNK1/JNK2/JNK3, 1:1000, #PA5-36,548, ThermoFisher; phospho-SAPK/JNK (Thr183/Tyr185) (81E11), 1:1000, #4668 Cell Signaling; Nrf2 1:500, #A1244, Abclonal; TGF-β1 1:500 # A18692; Smad3, 1:500, #A19115, Abclonal. The membranes were blocked with 2% BSA in Tris-buffered saline (TBS) and 0.01% Tween 20 overnight at room temperature and then incubated with primary antibody overnight at room temperature. Secondary antibody (1:10,000) was diluted in 2% BSA in TBS and 0.01% Tween 20, and membranes were incubated at room temperature for 1 h. Protein loading was assessed by immunoblotting using rabbit anti-actin, 1:10,000, Cell Signaling; mouse anti-GAPDH (0411), 1:5000, #sc-47724, SantaCruz; mouse anti-tubulin, 1:10,000, #AC030, Abclonal antibodies.
9
Immunohistochemical Profiling of Fibrosis Markers
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Antigen removal from tissue sections was performed using citrate buffer at 60 °C for 16 h. Immunohistochemistry (IHC) sections were treated for 15 min with hydrogen peroxide and blocked for 1 h using 1% goat serum. LF cells for immunofluorescence (IF) analysis were fixed in paraformaldehyde (4%), incubated in the presence of Triton-100 (0.5%) and blocked using bovine serum albumin (BSA, 5%) at room temperature for 2 h. Overnight incubation of the sections and cells at 4 °C was performed in the presence of primary antibodies against CLU (1:100, ABclonal; 1:100, Santa Cruz, USA), TGF-β1 (1:100, ABclonal), α-SMA (1:100, ABclonal), p-SMAD3 (1:100, Abcam), COL1A2 (1:100, ABclonal), vimentin (1:200, Santa Cruz), ALK5 (1:100, ABclonal), PRKD3 (1:100, Proteintech, China), and LAMP1 (1:50, Santa Cruz). Samples were washed, after which they were incubated with matched secondary antibody for 1 h. Hematoxylin (Beyotime) and DAB (Beyotime) stains were used for IHC, while DAPI stain was used to stain the nuclei during IF analysis.
10
Western Blot Analysis of EMT Markers
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The expression levels of GOLM1, E-cadherin, Vimentin, TGF-β1, Smad2, p-Smad2, and
GADPH were detected by Western blotting. First, equal amounts of proteins were
loaded onto 10% SDS-PAGE gels for separation and then transferred to
polyvinylidene fluoride membrane (Bio-Rad). Second, the membranes were blocked
with 5% nonfat milk for 2 h and then were incubated with primary antibodies, and
placed on shaker, at 4 °C overnight. Indicated primary antibodies in this study
were polyclonal rabbit antibodies against GOLM1 (1:1000 dilution; Affinity),
E-cadherin (1:5000 dilution; Proteintech), Vimentin (1:1000 dilution;
Proteintech), TGF-β1 (1:1000 dilution; Abclonal), Smad2 (1:1000 diluton;
Affinity), p-Smad2 (1:1000) and GADPH (1:1000 dilution; Affinity). Third, the
membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies
(1:600 dilution; Beyotime Biotechnology) after washing with TBST buffer.
Finally, the protein levels were detected by using an enhanced chemiluminescence
system (ECL Kit, Pierce Biotechnology) and captured on light-sensitive Xrayfilm
(Carestream). The optical densities of bands were analyzed by ImageJ
software.
GADPH were detected by Western blotting. First, equal amounts of proteins were
loaded onto 10% SDS-PAGE gels for separation and then transferred to
polyvinylidene fluoride membrane (Bio-Rad). Second, the membranes were blocked
with 5% nonfat milk for 2 h and then were incubated with primary antibodies, and
placed on shaker, at 4 °C overnight. Indicated primary antibodies in this study
were polyclonal rabbit antibodies against GOLM1 (1:1000 dilution; Affinity),
E-cadherin (1:5000 dilution; Proteintech), Vimentin (1:1000 dilution;
Proteintech), TGF-β1 (1:1000 dilution; Abclonal), Smad2 (1:1000 diluton;
Affinity), p-Smad2 (1:1000) and GADPH (1:1000 dilution; Affinity). Third, the
membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies
(1:600 dilution; Beyotime Biotechnology) after washing with TBST buffer.
Finally, the protein levels were detected by using an enhanced chemiluminescence
system (ECL Kit, Pierce Biotechnology) and captured on light-sensitive Xrayfilm
(Carestream). The optical densities of bands were analyzed by ImageJ
software.
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