Sodium butyrate (Purity≥98%) was purchased from Sigma Chemical (St. Louis, MO, USA). Docetaxel was purchased from Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China). Cell counting kit-8 was purchased from Dojindo Laboratories (Kumamoto, Japan). Hoechst 33258 and TUNEL assay were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Cell cycle staining kit was purchased from Multi Sciences Biotech Co., Ltd. (Hangzhou, China). Annexin V-APC/7-AAD apoptosis detection kit was purchased from BD Biosciences (San Diego, CA, USA). The primary antibodies against Gli1 and GAPDH were purchased from Abcam (Cambridge, MA, USA). The primary antibodies against Ki-67, CDK1, CDK2, Cyclin A, Cyclin D1, Bcl-2 and Bax were purchased from Wanlei Biotechnology Co., Ltd. (Shenyang, China). The primary antibodies against p21, cleaved-Caspase 3 and Survivin were purchased from Cell Signaling Technology (Danvers, MA, USA).
Cyclind1
Manufactured by Wanlei
Sourced in China, United States
CyclinD1 is a laboratory equipment product designed for the detection and analysis of cyclin D1 proteins. It is used in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Wanlei (6 mentions)
Bcl2 associated x (bax), by Wanlei (5 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Cyclina2, by Wanlei (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by Biosharp (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
β actin, by Boster Bio (4 mentions)
Protease inhibitor cocktail, by Beyotime (4 mentions)
Bcl 2, by Abcam (4 mentions)
E cadherin, by Wanlei (3 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
N cadherin, by Wanlei (2 mentions)
Vimentin, by Wanlei (2 mentions)
Beclin 1, by Wanlei (2 mentions)
β actin, by Wanlei (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
Annexin 5 apc 7 aad apoptosis detection kit, by BD (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
12 protocols using cyclind1
1
Sodium Butyrate Modulates Docetaxel Sensitivity
2
Immunoblotting Antibody Validation Protocol
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VP was purchased from Sigma (St Louis, MO). Antibody against PML/RARα was purchased from Abcam (China). Antibodies against MST1, LATS1, YAP, p-YAP (S127), p38 MAPK, ERK, p-ERK (Thr202/Tyr204), AKT, p-AKT (S473), p-S6 (S235/236), c-Myc and caspase3 were from Cell Signaling Technology (USA), Antibodies against PARP, Survivin, Bcl-2, Bax, connective tissue growth factor (CTGF) and cyclinD1 were from Wanleibio (China). The antibody against p-p38 MAPK (Thr180/Tyr182) was purchased from Millipore (USA). Goat anti-rabbit antibody, goat anti-mouse antibody and antibody against β-Actin were purchased from Zhongshan Goldenbrige Biotechnology Co., Ltd (China).
3
Comprehensive Cell Signaling Proteomics Protocol
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FN1 was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit primary antibodies Bax, Bcl-2, CyclinD1, E-cadherin, N-cadherin and enhanced chemiluminescence (ECL) kit were all obtained from Wanlei Biotechnology. Goat anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) and β-actin were purchased from DingguoChangsheng Biotechnology (Beijing, China). Fetal bovine serum (FBS), RPMI medium and Trizol reagent were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Propidium iodide (PI), dimethyl sulfoxide (DMSO), Triton X-100, RNAse, 4% paraformaldehyde and a bicinchoninic acid (BCA) protein assay kit were purchased from Sigma-Aldrich (Merck Kgaa, Darmstadt, Germany). Trypsin-EDTA (0.25%), penicillin/s-treptomycin (100×), tetramethylethylenediamine (TEMED), ammonium persulfate (AP), trihydroxymethyl aminomethane (Tris), glycine, Tween-20, sodium dodecyl sulfonate (SDS), apoptosis assay kit, RIPA lysis buffer and bovine serum albumin (BSA) were obtained from Genview Scientific, Inc. (El Monte, FL, USA). A PrimeScript® RT reagent kit and SYBR®Premix Ex TaqTM PCR kit were purchased from Takara Bio, Inc. (Otsu, Japan). Polyvinylidene difluoride (PVDF) membranes, 30% polyacrylamide were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).
4
Western Blot Analysis of Protein Expression
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The cells were collected, washed 3 times with cold PBS and treated with cold RIPA lysis buffer containing a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China). The BCA protein assay kit was used to detect the protein concentration, according to the manufacturer’s protocol (Beyotime Biotechnology, Shanghai, China). The extracted proteins were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature and then the following primary antibodies were added:β-Actin (Boster Biological Technology, CA, USA), MCM5 (Abcam, MA, UK), Bcl-2 (Abcam, MA, UK), CyclinD1 (Wanleibio, Shenyang, China), CyclinA2 (Wanleibio, Shenyang, China) and p53 (Wanleibio, Shenyang, China). This step was followed by the addition of horseradish peroxidase-conjugated secondary antibodies (1:4,000; Biosharp, China) and the signals were detected using an ECL (enhanced chemiluminescence) kit (Millipore, USA).
5
Protein Expression Analysis in Testicular Tissues
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Testicular tissues and GC-1 cells were lysed in RIPA buffer containing protease inhibitor from a protein extraction kit (Beyotime, China), and the BCA protein assay (Beyotime, China) was used to measure the protein concentration. Proteins were separated using 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 1 h at 37 °C with 5% dried fat-free milk in PBS-Tween 0.1% (PBST), and were incubated with specific primary antibodies against γ-H2AX (1:5,000), Cleaved Caspase3, p21, p-p53, p-CHK2, ATM (1:1,000, Cell Signaling Technology, Danvers, MA, USA), Bax, Bcl-2, CyclinD1 and CDK4 (1:500, Wanleibio, Shenyang, China), PCNA (1:5,000, Abcam Inc., Cambridge, MA, USA), PUMA, p-ATM, CHK2 (1:1000, BOSTER, Wuhan, China), p53, and β-actin (1:1,000, ZSGB-BIO, Beijing, China) overnight at 4 °C. The next day, the secondary antibodies (1:1,000, BOSTER, Wuhan, China) were incubated for 1 h at 37 °C with anti-mouse or anti-rabbit HRP-conjugated antibodies. Protein bands were detected using enhanced chemiluminescence (NCM Biotech, Suzhou, China), and the densities of those bands were quantified by ImageJ software.
6
Peiminine Modulates Autophagy and Apoptosis
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Antibodies targeting the following proteins were used in this study: cyclin D1 (WL01435a), CDK2 (WL01543), p27 (WL04174), cleaved caspase-9 (WL01838), cleaved caspase-3 (WL01992), Bcl-2 (WL01556), Bax (WL01637), LC3BII/I (WL01506), beclin-1 (WL02508), p62 (WL02385), p-JNK (WL01813), JNK (WL01295), c-JUN (WL02863), E-cadherin (WL01482), vimentin (WL01960), MMP-2 (WL1579), MMP-9 (WL01580), β-actin (WL01372), goat anti-rabbit IgG-HRP (WLA023) (all from Wanleibio, China), and p-c-JUN (AF3095, Affinity, United States). Purified peiminine (98%) was obtained from Yuanye Bio Co., Ltd (Shanghai, China; B20081). Cell Counting Kit-8 (CCK-8; C0037), the live/dead assay kit, the cell cycle and apoptosis analysis kit, the ROS Kit, NAC, SP600125, and the terminal dUTP nick-end labeling (TUNEL) Kit (C1086) were acquired from Beyotime Institute of Biotechnology (Shanghai, China). Tetramethylrhodamine methyl ester (TMRM; I34361) and Hoechst 3342 (H3570) were sourced from Life Technology (OR, United States). The mRFP-GFP-LC3 adenovirus was acquired from HanBio Technology Co., Ltd (Shanghai, China). The rest of the reagents and experimental materials were purchased from common commercial sources.
7
Western Blot Analysis of Cell Cycle and Autophagy Markers
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FA was purchased from Meilunbio (Dalian Meilun Biotechnology Co., LTD. Liaoning, China). Antibodies for P53, P21, Cyclin D1, Cyclin E, Beclin-1, LC3-II, Atg12-Atg5 and β-actin used for Western blot analysis were purchased from Wanleibio (Shenyang, Liaoning, China). Super moloney-murine leukemia virus (M-MLV) reverse transcriptase for fluorescence quantification was purchased from BioTeke (Beijing, China) and RNA simple Total RNA Kit was purchased from TIANGEN (Beijing, China).
8
Protein Extraction and Western Blot Analysis
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Protein was extracted using RIPA buffer containing 1 % PMSF (SolarBio, Beijing, China). A BCA kit (Beyotime, Shanghai, China) was used to determine the protein concentration. The isolated proteins were transferred to polyvinylidene fluoride membranes after 10 % SDS polyacrylamide gel electrophoresis. The membranes were incubated overnight with primary antibodies to cyclin D1 (Wanlei, Shenyang, China), PCNA (Wanlei, Shenyang, China), CDK2 (Abcam Ltd., Cambridge, MA, USA), MyoD (Abcam Ltd., Cambridge, MA, USA), myogenin (MyoG; Abcam Ltd., Cambridge, MA, USA), MyHC (Genetex, Southern California, USA) and β-actin (Abcam, Cambridge, UK) at 4 °C. The membranes were then incubated with the corresponding secondary antibodies at room temperature for 1 h. After washing, the membranes were exposed to ECL Plus (SolarBio, Beijing, China) and the Chemidoc XRS+ system (Bio-Rad, California, USA) was used for imaging the resultant bands.
9
Western Blot Analysis of Signaling Pathways
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Cell was lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethanesulfonyl fluoride, and protein concentrations were detected using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai) according to the manufacturer’s protocol. The proteins were then separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked in 5% milk for 2 h at room temperature and incubated with antibodies for OSR1 (Santa Cruz Biotechnology, sc-376545, 1:1000), FAK (Santa Cruz Biotechnology, sc-271126, 1:1000), p-FAK (Tyr397) (Santa Cruz Biotechnology, sc-81493, 1:1000), AKT (Wanleibio, WL0003b, 1:500), p-AKT (Ser473) (Wanleibio, WLP001a, 1:500), p38 (Wanleibio, WL00764, 1:500), p-p38 (Thr180/Tyr182) (Wanleibio, WLP1576, 1:500), ERK 1/2 (Cell Signaling Technology), p-ERK1/2 (CST), E-cadherin (Wanleibio, WL01482), N-cadherin (Wanleibio, WL01047), Vimentin (Wanleibio, WL01960), p21 (Wanleibio, WL0362), Cyclin D1 (Wanleibio, WL01435a), Snail (Wanleibio, WL01863), c-Myc (Wanleibio, WL01781), Bcl-2 (Wanleibio, WL01556), and GAPDH (BOSTER, BM1623, 1;2000) at 4°C overnight. Then, the blots were washed and incubated with second antibodies (1:5000, Bioss) for 1 h at room temperature. Finally, the blots were washed and visualized with enhanced chemiluminescence (ECL) reagents (Wanleibio, Shenyang).
10
Protein Expression Analysis Pipeline
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The cells were collected, washed 3 times with cold PBS and treated with cold RIPA lysis buffer containing a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China). The BCA protein assay kit was used to detect the protein concentration, according to the manufacturer's protocol (Beyotime Biotechnology). The extracted proteins were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature and then the following primary antibodies were added:β-Actin (BOSTER Biological Technology, CA, USA), MCM5 (Abcam, MA, USA), Bcl-2 (Abcam, MA, USA), CyclinD1 (Wanleibio, Shenyang, China), CyclinA2 (Wanleibio, Shenyang, China) and p53 (Wanleibio, Shenyang, China). This step was followed by the addition of horseradish peroxidase-conjugated secondary antibodies (1:4,000; Biosharp, China) and the signals were detected using an ECL (enhanced chemiluminescence) kit (Millipore, USA).
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