Total proteins were obtained from HPASMCs and human pulmonary arteries. The concentration of proteins was assessed with a BCA kit (Aspen, Wuhan, China). Whole lysates were isolated by SDS-PAGE and transferred to PVDF membranes that were blocked by TBST with 5% non‐fat dry milk at room temperature for 1h. The membranes were first incubated with primary antibodies at 4°C overnight and then HRP-conjugated secondary antibodies at room temperature for 1h. The western ECL substrate (Bio-Rad, California, USA) and the ChemiDocTM-XRS+imaging system (Bio-Rad, California, USA) were used to visualize the bands. ImageJ software (NIH, Bethesda, MD) was used to measure the band intensities quantitatively. The following primary antibodies were used: against β-ACTIN (Sungene Biotech, Tianjin, China), GAPDH (Sungene Biotech, Tianjin, China), MBD2 (Abcam, Cambridge, UK), Cyclin D1 (Cyclin D1, Proteintech, Wuhan, Hubei, China), PCNA (Proteintech, Wuhan, Hubei, China), and BMP2 (Proteintech, Wuhan, Hubei, China).
Cyclin d1
Manufactured by Proteintech
Sourced in United States, China, United Kingdom
Cyclin D1 is a protein involved in regulating the cell cycle. It plays a critical role in the G1/S transition, promoting the progression of cells from the G1 phase to the S phase of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (40 mentions)
Bcl 2, by Proteintech (27 mentions)
Bcl2 associated x (bax), by Proteintech (26 mentions)
E cadherin, by Proteintech (24 mentions)
Pvdf membrane, by Merck Group (23 mentions)
C myc, by Proteintech (19 mentions)
N cadherin, by Proteintech (19 mentions)
β actin, by Proteintech (19 mentions)
Cyclin e1, by Proteintech (17 mentions)
β catenin, by Proteintech (15 mentions)
Cyclin b1, by Proteintech (15 mentions)
Vimentin, by Proteintech (14 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (11 mentions)
Cyclin a2, by Proteintech (10 mentions)
E cadherin, by Cell Signaling Technology (9 mentions)
P akt, by Cell Signaling Technology (9 mentions)
P akt, by Proteintech (8 mentions)
Bcl 2, by Cell Signaling Technology (8 mentions)
β catenin, by Cell Signaling Technology (8 mentions)
158 protocols using cyclin d1
1
Western Blot Analysis of Pulmonary Proteins
2
Western Blot Analysis of Protein Targets
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Cells were lysed in RIPA buffer (Pioneer, Shanghai, China) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (TargetMol, USA). Protein concentration determined using a BCA protein assay kit (Proteintech, Wuhan, China). The protein samples were transferred to PVDF membrane by SDS-PAEG, membranes were incubated with specific primary antibody at room temperature for 30 min and then overnight at 4 °C. Then they were incubated with corresponding anti-rabbit/anti-mouse secondary antibody (TransGen Biotech, Beijing) at room temperature for 1 h. The membranes were incubated with ECL (Boster Biological Technology co.ltd, California, USA) in the dark for chemiluminescence detection. Luminescent signals were detected and recorded by Syngene GBox (Syngene, Cambridge, UK). The primary and secondary antibodies used are listed as follows: RPL5 (Proteintech, 29,092–1-AP), MMP2 (Proteintech, 10,373–2-AP), MMP9 (Proteintech, 10,375–2-AP), CDK4 (Proteintech, 11,026–1-AP), CyclinD1 (Proteintech, 26,939–1-AP), MEK1/2 (Proteintech, 11,049–1-AP), p-ERK (Proteintech, 28,733–1-AP), ERK (Proteintech, 67,170–1-Ig), c-Myc (Proteintech, 10,828–1-AP), p-MEK1/2 (Cell Signaling Technology, 9154), FOXO3 (Boster, BM4734) and β-Tubulin (Proteintech, 66,240–1-Ig).
3
Stem Cell Differentiation Protocol
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Fentanyl citrate was obtained from Northeast Pharmaceutical Group (China). The sources of reagents were as follows: Sox2, Oct4, N-cadherin, Vimentin, E-cadherin, β-catenin, GSK-3β, CD44, Cyclin D1, and GAPDH antibody (Proteintech, China); Nanog antibody (Abcam, USA); FUT8, c-Jun and VEGF antibody (Santa Cruz, USA); Lens culinaris agglutinin (LCA) Lectin, binding of α1, 6-fucosylation epitope. (Vector Laboratories, USA); p-GSK-3β (Ser9) antibody (Elabscience, China); LGK-974 (inhibitor of Wnt ligands) (Selleck, USA).
4
Western Blot Analysis of JAK-STAT Pathway
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Differentiated cells were harvested and lysed in RIPA buffer supplemented with proteinase inhibitor cocktail (MedChemExpress). Proteins were separated on 10% SDS-PAGE gel and electro-transferred to PVDF membranes (Millipore). The membranes were blocked in blocking solution (5% skim milk in PBS) for 1 h at room temperature, and incubated overnight at 4°C with primary antibodies against JAK2 (1:1000, Cell Signaling Technology), p-JAK2 (Tyr1007/1008, 1:1000, Abcam), STAT3 (1:2000, Cell Signaling Technology), p-STAT3 (Tyr705, 1:2000, Abcam), HIF-1a (1:500, Santa Cruz Biotechnology), cyclin D1 (1:5000, proteintech), c-Myc (1:1000, Abmart), GAPDH (1:2000, Affinity) and β-actin (1:3000, Affinity). Thereafter, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-linked secondary antibodies (1:2000, Wanleibio). The protein bands were detected by using an enhanced chemiluminescence kit (Wanleibio).
5
Citrus Peel Extract Modulates Cell Signaling
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The dried citrus peel was purchased from the Jiangsu Provincial Hospital of Traditional Chinese Medicine (Nanjing, China). The CAE was then prepared as previously described. Dickkopf-1 (DKK1) was purchased from R&D Systems (USA); dimethyl sulfoxide, fetal bovine serum, Dulbecco’s modified Eagle’s medium and trypsin were purchased from Gibco, Thermo Fisher Scientific-CN (Shanghai, China); penicillin and streptomycin were purchased from Biological Industries (HaEmek, Israel); platelet-derived growth factor a (PDGF-a), PDGF-b, tumor necrosis factor α (TNF-α), matrix metalloproteinases-7 (MMP-7), CyclinD1, connective tissue growth factor (CTGF), α-smooth muscle aorta (a-SMA), collagen I, collagen III, β-catenin, glycogen synthase kinase-3β (GSK-3β), β-actin, β-tubulin, and GAPDH were purchased from Proteintech (Wuhan, China); and p53, p21, and p16 were purchased from Abcam (Cambridge, UK).
6
Exosome Protein Analysis by Western Blot
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Cells or purified Exo samples were diluted 1:5 with protein loading buffer (6 ×) (Transgen Biotech, Beijing, China) and heated at 99 °C for 10 min. Protein extracts were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and transferred onto polyvinylidene difluoride membranes (Sigma Aldrich Chemie GmbH, Munich, Germany) at 100 V for 30–60 min. The membranes were blocked with 5% nonfat milk at 23–25 °C for 1 h, washed three times in TBST buffer for 10 min, and incubated with the following primary antibodies at 4 °C overnight: CD9 (1:1000; BioLegend, San Diego, CA, USA), Alix (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Cyclin A2 (1:1000; Proteintech Group, Rosemont, IL, USA), Cyclin D1 (1:5000; Proteintech), VEGFA (1:1000; Proteintech), CXCL12 (1:500; Proteintech), and GAPDH (1:5000; Proteintech). GAPDH was used as a loading control. Western blots were probed with IRDye 800-conjugated goat anti-rabbit or anti-mouse secondary antibodies and blotted proteins detected using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).
7
Investigating Icaritin's Anticancer Effects
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Icaritin was purchased from Sigma-Aldrich (MO, United States). Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), crystal violet, a cell-based ROS assay kit, N-acetylcysteine (NAC), and a senescence-associated β-galactosidase (SA-β-Gal) kit were obtained from Beyotime Biotechnology (Beijing, China). Annexin V-FITC/PI and propidium iodide (PI) were purchased from KeyGEN BioTECH (Nanjing, China). The primary antibody against γ-H2AX was obtained from Cell Signaling Technology (MA, United States). Primary antibodies against ki67, GAPDH, PARP1, cyclin D1, CDK2, CDK4, Bax, Bcl-2, P53, P21, p-EGFR, EGFR, p-AKT, AKT, and HIF1α were obtained from Proteintech (Rockville, MD, United States). Antibody against horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-mouse or goat-anti-rabbit) were obtained from ZSGB-BIO (Beijing, China).
8
Western Blot Analysis of Protein Expression
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Cell samples were washed with ice-cold PBS and then lysed by RIPA (Beyotime, China) containing protease inhibitors (Beyotime, China). Cell protein lysates were separated in 10 % SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were blocked by 5 % skim milk soluted in TBST buffers, and were incubated with primary antibodies for ADAM10, c-myc (Abcam, UK), E-cadherin, MMP9 (Santa Cruz Biotechnology, USA), β-catenin and cyclinD1 (ProteinTech Group, USA) overnight at 4 °C. PVDF membranes were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (ProteinTech Group, USA). Antibody against GAPDH (Cell Signaling Technology, USA) was used as an internal control. Antibody against Histone H3 (Abcam, UK) was used as an internal control for nuclear β-catenin. The protein of interest was visualized using ECL Western blotting substrate (Pierce, USA).
9
Molecular Mechanisms of Fibrosis Inhibition
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Sodium hydrogen sulfide (NaHS), PPG (a CSE inhibitor) and 7-Azido-4-Methylcoumarin (AzMC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). SKF38393 (a DR1 agonist) was obtained from Abcam (Cambridge, MA, USA). Y-27632 (a ROCK inhibitor) was obtained from MedChemExpress (Shanghai, China). The primary antibodies for anti-CSE, Cyclin D1, proliferating cell nuclear antigen (PCNA), p21 Cip/W AF -1 , collagen I (Col-1), collagen III (Col-3), matrix metalloproteinase 9 (MMP-9), osteopontin (OPN) and α-smooth muscle actin (α-SMA) were purchased from Proteintech (Wuhan, China). The anti-p-RhoA, t-RhoA, p-ROCK1, t-ROCK1 were from Affinity Biosciences (Cincinnati, OH, USA). The anti-DR1 antibody was from GeneTex (Irvine, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG antibody, the Cell Counting Kit-8 (CCK-8) and anti-β-actin were obtained from Boster Bioengineering Limited Company (Wuhan, China). The EdU Cell Proliferation Assay Kit were obtained from Ribobio (Guangzhou, China). Fluo-4 AM were obtained from Beyotime Biotechnology (Shanghai, China). Enhanced ECL Chemiluminescent Substrate Kit was obtained from Yeasen Biotechnology (Shanghai, China). All other chemicals were from Solarbio (Beijing, China) or Beyotime Biotechnology.
10
Western Blot Analysis of Cellular Proteins
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The cell lysates were prepared as previously described,[28 ] and the protein concentration was determined with a BCA kit (Thermo Fisher Scientific). Protein samples were separated by sodium dodecyl sulfate (SDS)‐PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% fat‐free milk for 1 h, the PVDF membrane was incubated with primary antibody for 1–2 h at room temperature and then washed with 1× tris‐buffered saline with Tween (TBST) for 30 min. Next, the membrane was incubated with the corresponding horseradish peroxidase‐conjugated secondary antibodies for 1 h. After washing with TBST, signals were detected using ECL substrate (Bio–Rad, Hercules, CA, USA). The antibodies used included actin and RanBP3 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CDK4 and p‐Rb from Cell Signaling Technology (Beverly, MA, USA), cyclin D1, CDK6, GAPDH, histone H3, lamin B1, β‐catenin, and c‐Myc from Proteintech (Rosemont, IL, USA).
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