The drug-treated cells were lysed with RIPA lysis buffer (Genestar, Taipei, Taiwan) and then separated by SDS—PAGE before transfer to polyvinylidene difluoride (PVDF) membranes. The membranes were immersed in a blocking buffer (5% nonfat milk in TBS containing 0.1% Tween-20) for 2 h at room temperature with gentle agitation. Sequentially, the membranes incubation with primary antibodies against Collagen Type I (Proteintech, Rosemont, USA), PAI-1 (Cell Signaling, Massachusetts, USA), CTGF (Proteintech), Beclin 1 (Cell Signaling), p62 (MBL, Woburn, USA), LC3 (Cell Signaling), NLRP3 (Proteintech), ASC (AdipoGene, San Diego, USA), caspase 1 (Proteintech), and GAPDH (Proteintech) overnight at 4°C. After washing three times with TBS-T at room temperature, the membranes were incubated with secondary antibodies for 1 h at room temperature and washed with TBS-T three times. Immunoreactive bands were visualized by a chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA).
Collagen type 1
Manufactured by Proteintech
Sourced in China, United States
Collagen Type I is a purified collagen protein derived from bovine sources. It is a key structural component of various connective tissues in the body, including skin, bone, and cartilage. This product is suitable for use in cell culture, tissue engineering, and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
E cadherin, by Proteintech (2 mentions)
α sma, by Proteintech (2 mentions)
Chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Caspase 1, by Proteintech (1 mentions)
Pai 1, by Cell Signaling Technology (1 mentions)
Nlrp3, by Proteintech (1 mentions)
Fibronectin, by Proteintech (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Pierce bca protein assay kit, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab3442, by Abcam (1 mentions)
Ab84593, by Abcam (1 mentions)
Ab64943, by Abcam (1 mentions)
Ab64644, by Abcam (1 mentions)
9 protocols using collagen type 1
1
Western Blot Analysis of Autophagy and Inflammation Markers
2
Protein Expression Analysis of Frozen Lung Tissues
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Proteins of the frozen lung tissues were lysed on ice in RIPA buffer (Beyotime, Shanghai, China) containing proteinase inhibitors (Meilunbio, Dalian, China). The total protein content was evaluated using a Pierce BCA Protein Assay Kit (Beyotime, Shanghai, China). The total proteins were electrophoretic separated and transferred on PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking for 1 h with bovine serum albumin, the membranes were incubated at 4 °C overnight with primary antibody: Fibronectin (Proteintech, Wuhan, China, 1:1000), Collagen Type I (Proteintech, Wuhan, China, 1:1000), cleaved caspase-3 (Abcam, Cambridge, MA, USA, 1:500), Bax (Cell Signaling, BSN, USA, 1:1000), and GAPDH (Abcam, Cambridge, MA, USA, 1:10,000). After washing with TBST, the membranes were incubated with anti-rabbit IgG secondary antibody (Elibscience, China, 1:2000). The proteins bands were visualized using ECL detection reagents (Shandong Sparkjade Biotechnology Co., Ltd., Jinan, China) and analyzed in chemiluminescence imaging analysis system (Sagecreation, Beijing, China). Densitometry analysis of western blot was quantified via Image J software.
3
Protein Expression Analysis in Cardiac Tissue
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Apical myocardia harvested from four to six hearts per treatment group were homogenized in a lysis buffer containing phosphatase and proteinase cocktail inhibitor, in a ratio of 100:1:1, respectively. Lysates of normalized concentrations treated with reducing agents were denatured at 100°C for 10 min and separated on SDS-PAGE gels. The transferred protein bands were blocked and immunoblotted overnight in the following primary antibodies: β1AR (ab3442; Abcam), β2AR (ab182136; Abcam), MEF2 (ab64644; Abcam), GRK5 (ab64943; Abcam) GATA4 (ab84593; Abcam), NFAT (ab25916; Abcam), AC5 (PAC-501AP; FabGennix), AC6 (PAC-601AP; FabGennix), AC7 (PAC-701AP; FabGennix), ANP (sc-515701; Santa Cruz Biotechnology), BNP (sc-271185; Santa Cruz Biotechnology), GRK2 (sc-13143; Santa Cruz Biotechnology), pNF-κB (3033T; Cell Signaling Technology), NF-κB (8242T; Cell Signaling Technology), Cleaved Caspase-3 (9661T; Cell Signaling Technology), Collagen Type I (14695-1-AP, Proteintech), Collagen Type III (13548-1-AP, Proteintech), and GAPDH (10494-1-AP; Proteintech). Immunoblots were performed in triplicates and normalized with their respective loading controls.
4
Quantifying Myocardial Protein Markers
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Concentrations of lysates obtained from apical myocardia (n = 4 hearts per group) were normalized, treated with loading buffer, and proteins denatured at 100°C for 10 min. The proteins were separated were SDS-PAGE gels, transferred on PVDF membranes which were blocked and blotted overnight with the following antibodies: Collagen Type I (Proteintech; 14695-1-AP), Collagen Type III (Proteintech; 13548-1-AP), ANP (Santa Cruz Biotechnology; sc-515701), BNP (Santa Cruz Biotechnology; sc-271185), Cleaved Caspase-3 (Cell Signaling Technology; 9661T) and GAPDH (Proteintech; 10494-1-AP). Western blots were performed in triplicates and normalized with their respective loading controls.
5
Western Blot Analysis of Protein Markers
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Cell and tissue protein was extracted using RIPA buffer (Beyotime, China). Proteins (20-30 mg) were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis gel before being transferred to polyvinylidene difluoride membranes (Merck Millipore, USA). After being blocked in 5% milk for 1 h, the membranes were then probed at 4°C overnight with antibodies against PTEN (1:500; Santa Cruz Biotechnology, USA, sc-7974), AKT (1:1000; Proteintech Group, USA, 10176-2-AP), p-AKT (1:1000; Proteintech Group, USA, 66444-1-lg), HIF-1α (1:1000; Proteintech Group, USA, 20960-1-AP), MMP9 (1:1000; Proteintech Group, USA, 10375-2-AP), Collagen Type I (1:1000; Proteintech Group, USA, 14695-1-AP), E-cadherin (1:1000; Proteintech Group, USA, 20874-1-AP), Smooth Muscle Actin (1:1000; Proteintech Group, USA, 14395-1-AP), CD63 (1:1000; Proteintech Group, USA,25682-1-AP), CD81 (1:1000; Proteintech Group, USA, 66866-1-lg), HSP70 (1:1000; Abcam , USA, ab5439), β-Tubulin (1:5000; Cell Signaling Technology, USA, 2146) and GAPDH (1:5000; Abcam , USA, ab8245). The membranes were washed by TBS with Tween, then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. After that, the membranes were immersed in ECL (Abbkine, China) and visualized using a chemiluminescence instrument.
6
Immunohistochemical Analysis of Smooth Muscle and Collagen
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The paraffin sections were successively deparaffinized with xylene and dehydrated with ethanol. After repairing the antigen with microwave, membranes were broken with Triton X-100 (Servicebio), then incubated with goat serum after blocking endogenous peroxidase. They were subsequently incubated with monoclonal antibodies to smooth muscle actin (Proteintech, Wuhan, China) or collagen type I (Proteintech) overnight at 4 ℃. The samples were incubated with a goat anti-rabbit IgG monoclonal antibody (Proteintech) for 1 h at room temperature. The nuclei were stained with hematoxylin after staining with DAB chromogenic solution.
7
Effects of Taurocholate on LX-2 and HepG2 Cells
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LX-2 cells were treated with Na+/taurocholate (Sigma) (50 μM and100μM) dissolved in DMEM without FBS for 24 h. LX-2 cells co-cultured with HepG2 cells were also treated with Na+/taurocholate (Sigma) (50 μM and 100 μM) dissolved in DMEM without FBS for 24 h. Total cellular proteins were extracted with a protein exaction kit (Beyotime Biotechnology) according to the manufacturer’s protocols. BCA Protein Assay Kit (Beyotime Biotechnology) was used for quantification of exacted proteins. After separated with SDS-PAGE, the proteins were transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TTBS). The membranes were then incubated with the primary antibodies against Toll-like receptor4 (TLR4) (Santa Cruz Biotechnology), alpha Tubulin (α-Tubulin) (Proteintech), Collagen Type I (Proteintech) and smooth muscle actin (α-SMA) (Proteintech) overnight at 4 °C, and subsequently incubated with the secondary antibody for 2 h at 37 °C. Protein band was detected with the enhanced chemiluminescence (Advansta) method and imaged with a Bio-Rad ChemiDoc MP imaging system. Intensity of the α-Tubulin band was as the internal reference.
8
Comprehensive Protein Expression Analysis
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The antibodies of Smad2/3 (3102, CST), AKT (#9272, CST), p-AKT (#9271, CST), p-Smad2/3 (#ab272332, Abcam), E-cadherin (#20874-1-AP, Proteintech), N-cadherin (#22018-1-AP, Proteintech), Vimentin (#BF8006, Affinity Biosciences), Collagen Type I (#14695-1-AP, Proteintech), NF-κB1 (#14220-1-AP, Proteintech), LEF-1 (#28540-1-AP, Proteintech), PD-L1 (#66248-1-Ig, Proteintech), GAPDH (#60004-1-Ig, Proteintech), SNAIL (#AF6032, Affinity Biosciences), ROCK1 (#PTM-6454, PTM Bio), ROCK2 (#PTM-6169, PTM Bio), Ki67 (#GB111499, Servicebio), and fibrillar actin (F-actin) (#C1033, Beyotime) were used for western blot, immunofluorescence staining, and immunohistochemical staining. The following antihuman antibodies were used for flow cytometry: CD45 (PE, 30-F11; eBioscience), CD8α (APC, 53-6.7; eBioscience), 7-AAD (Invitrogen), CD3 (eFluor™ 506, UCHT1; eBioscience), PD-1 (Pacific Blue, EH12.2H7; Biolegend), and TIM-3 (FITC, F38-2E2; Biolegend).
9
Protein Expression Analysis by Western Blot
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Tissues or cells were harvested at the experimental endpoint and lysed with RIPA buffer (Beyotime, Nantong, China) with 1 mM PMSF (ST505, Beyotime. Nantong, China). A bicinchoninic acid protein assay kit (Thermo Fisher, Waltham, MA, USA) was used to measure the protein concentration of each sample.
Equal amounts of proteins were separated by SDS-PAGE and transferred onto PVDF membranes. Then, the PVDF membranes were blocked using 5% milk for 2 h and subsequently incubated with primary antibodies overnight at 4 °C. The following primary antibodies were utilized: β-tubulin (1:1,000, Bioworld Technology, Minnesota, USA), GAPDH (1:1,000, Kangchen, Shanghai, China), α-SMA (1:1,000, Proteintech Group, Wuhan, China), Collagen type I (1:1,000, Proteintech Group, Wuhan, China), Bax (1:1000, Cell Signaling Technology, Boston, Massachusetts, USA), Bcl-2 (1:1,000, Cell Signaling Technology, Boston, Massachusetts, USA), and PPARγ (1:1,000, Proteintech Group, Wuhan, China). β-tubulin or GAPDH was used as a loading control. On the second day, the membranes were incubated in the appropriate secondary antibodies for 2 h at room temperature, and then the signals were visualized with an ECL Chemiluminescence Kit (Thermo Fisher). Analysis of each band was carried out with ImageJ software (National Institutes of Health).
Equal amounts of proteins were separated by SDS-PAGE and transferred onto PVDF membranes. Then, the PVDF membranes were blocked using 5% milk for 2 h and subsequently incubated with primary antibodies overnight at 4 °C. The following primary antibodies were utilized: β-tubulin (1:1,000, Bioworld Technology, Minnesota, USA), GAPDH (1:1,000, Kangchen, Shanghai, China), α-SMA (1:1,000, Proteintech Group, Wuhan, China), Collagen type I (1:1,000, Proteintech Group, Wuhan, China), Bax (1:1000, Cell Signaling Technology, Boston, Massachusetts, USA), Bcl-2 (1:1,000, Cell Signaling Technology, Boston, Massachusetts, USA), and PPARγ (1:1,000, Proteintech Group, Wuhan, China). β-tubulin or GAPDH was used as a loading control. On the second day, the membranes were incubated in the appropriate secondary antibodies for 2 h at room temperature, and then the signals were visualized with an ECL Chemiluminescence Kit (Thermo Fisher). Analysis of each band was carried out with ImageJ software (National Institutes of Health).
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