Baicalin (98%), with batch number YS121121, was purchased from the Xi'an Yuansen Biotechnology Company. 3-MA and LC3-II, LC3-I, Beclin-1, P62, p-AMPK, AMPK, mTOR, and tubulin monoclonal antibodies were purchased from Affinity Biosciences (Cincinnati, OH, USA). The CCK-8 kit, RIPA lysis buffer, polycarbonate Lucifer Yellow (dextran) and the reverse transcription kit were purchased from Sparkjade (Jinan, China). Bafilomycin A1 was purchased from BioVision Incorporated (Waltham, MA, USA). The Caco2 cell line was obtained from the Chinese Academy of Sciences (Beijing, China). The Millicell system and fluorescence plate reader were obtained from Sparkjade (Jinan, China).
Beclin 1
Manufactured by Affinity Biosciences
Sourced in United States, China
Beclin-1 is a core autophagy-related protein that plays a crucial role in the initiation and regulation of the autophagy process. It is essential for the formation of the autophagosome, a key structure involved in the degradation and recycling of damaged or unwanted cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Affinity Biosciences (3 mentions)
β actin, by Affinity Biosciences (2 mentions)
Reverse transcription kit, by Sparkjade (1 mentions)
Ripa lysis buffer, by Sparkjade (1 mentions)
P ampk, by Affinity Biosciences (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
P akt, by Affinity Biosciences (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Beyotime (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Cell cycle analysis kit, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Solarbio (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Trypsin, by Solarbio (1 mentions)
Cyclin d1, by Affinity Biosciences (1 mentions)
E cadherin, by Affinity Biosciences (1 mentions)
Vimentin, by Affinity Biosciences (1 mentions)
Hoechst 33258, by Beyotime (1 mentions)
9 protocols using beclin 1
1
Baicalin Modulates Autophagy Pathway
2
Quantitative Protein Expression Analysis
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The total proteins were extracted on ice from the cells and liver tissues by ultrasonic lysis in RIPA buffer containing 1 mM PMSF and 1% phosphatase inhibitor cocktail. The lysates were centrifuged at 12000 ×g for 15 min at 4 °C. The protein concentration in the supernatant of the lysates was determined with a BCA protein quantitative kit. Next, the samples were normalized, mixed with loading buffer, and denatured by heating at 95 °C for 5 min. Aliquots of 50 mg of proteins were loaded onto 8, 10, or 12.5% SDS-PAGE gels. The proteins were transferred onto 0.45 μm PVDF membranes. The membranes were blocked and incubated overnight at 4 °C with primary monoclonal antibodies against SIRT-1, PGC-1α, Cytochrome C oxidase IV (COX-IV), PPAR-ɑ, Mitochondrial fission factor (MFF), Mitofusin-1 (MFN1), p62/SQSTM1 (p62), Light chain 3B (LC3B), Beclin-1, and GAPDH (all from Affinity Biosciences, OH, USA). The membranes were washed with TBST and incubated with secondary antibodies at room temperature for 40 min, and after washing them five times, an enhanced chemiluminescence kit was used to detect the proteins on the membranes. The bands were quantified using the ImageJ system (v1.8.0, NIH, Bethesda, USA).
3
Western Blot Analysis of Cerebral Cortex
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Western blot assay was conducted following the standard protocol [31 ]. The brain samples of left cerebral cortical tissues were collected from TEIH rats, and were homogenized in RIPA and were then centrifuged at 10,000 r per min for 30 min. We mixed the supernatant with the loading buffer and then boiled it for 10 min before use. 50 μl protein was loaded into each lane of the SDS-PAGE gel for electrophoresis. The separated proteins were then transferred onto nitrocellulose membranes, which were blocked by using 5 % non-fat milk in TBST for 1 h at RT, and were then incubated overnight, separately with the following primary antibodies at 4 °C: LC3 (#AF5402, Affinity, USA), BECLin-1 (#AF5128, Affinity, USA), Akt (#AF6261, Affinity, USA), p-Akt (#AF0016, Affinity, USA) at a 1:10000 dilution ratio and β-actin (#AF7018, Affinity, USA) at a dilution ratio of 1:5000. After that, the PVDF membrane was washed using TBST for 15 min in triplicate for the incubation with HRP-conjugated secondary antibody (1:10000, CST, USA). Finally, the membranes were washed 20 min with TBST. The bands of interested protein were visualized using ECL (Merck Millipore, USA) and were exposed on the X-ray film. The intensity of the signals was quantified by using Image J software.
4
Neoprzewaquinone A Cytotoxicity Evaluation
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Neoprzewaquinone A (purity ≥98%) was obtained from Herbpurify (Chengdu, China). SGI-1776 and Netarsudil (purity ≥98%) were purchased from Selleck (Chengdu, China). RPMI 1640, DMEM, DMEM/F12 media and Fetal Bovine Serum (FBS) were acquired from Gibco (NewYork, NY, USA). Paraformaldehyde (PFA; 4%) was purchased from Biosharp (Hefei, China). The BCA protein assay kit, cell cycle analysis kit, annexin V-FITC/PI apoptosis detection kit and Hoechst 33258 were obtained from Beyotime (Shanghai, China). The trypsin, crystal violet (1%), and Mondansylcadaverine (MDC) assay kit were purchased from Solarbio (Beijing, China). The primary antibodies against ROCK1, ROCK2, mTOR/p-mTOR, MYPT1/p-MYPT1, PIM1, BAD/p-BAD, STAT3/p-STAT3, E-cadherin, Vimentin, cyclin B1, cyclin D1, Beclin1, ATG5, LC3B, GAPDH, β-actin, and HRP-conjugated secondary antibodies were purchased from Affinity (Jiangsu, China). The electrochemiluminescence (ECL) western blot detection kit was purchased from Abbkine (Redlands, CA, USA).
5
Molecular Mechanisms of Autophagy Regulation
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EDA (purity = 99.59%), 3‐Methyladenine (3‐MA, purity = 99.83%) and Rapamycin (Rapa, purity = 99.77%) were purchased from MedChem Express (New Jersey, US). Primary antibodies against transforming growth factor‐β1 (TGF‐β1), vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9) were purchased from Wanleibio (Shenyang, China). Heme oxygenase‐1 (HO‐1), NAD(P)H quinone oxidoreductase 1 (NQO1) and β‐actin antibodies were purchased from ABclonal Technology (Wuhan, China). LC3, Beclin1, p62 and Atg5 antibodies were purchased from Affinity Biosciences (OH, US). Cleaved‐Caspase3, Bcl‐2, Bax, p‐PI3K, PI3K, p‐AKT, AKT, p‐mTOR and mTOR antibodies were purchased from Cell Signalling Technology (Danvers, MA). RPMI 1640 medium was purchased from Gibco (Grand Island, NY) and fetal bovine serum was purchased from Animal Blood Ware (Shanghai, China).
6
Biochemical Assays for Cellular Stress
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Assay kits for detecting BUN, CREA, tumor necrosis factor-α (TNF-α), interleukin (IL)-8, reactive oxygen species (ROS) and superoxide dismutase (SOD) were purchased form Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Antibodies LC3, Beclin 1, AMPK, phosphorylated-AMPK (p-AMPK, Thr172), mTOR, phosphorylated-mTOR (p-mTOR, Ser2448), cleaved caspase 3, pro-caspase-3, Bcl-2, Bax and glyceraldehyde-3-phosphate dehydrogenase (GADPH) were ordered from Affinity (Changzhou, China). DB was obtained from ChemFaces (Wuhan, China). Compound C (CC), the specific AMPK signaling blocker, was purchased from MedChemExpress (Shanghai, China).
Antibodies LC3, Beclin 1, AMPK, phosphorylated-AMPK (p-AMPK, Thr172), mTOR, phosphorylated-mTOR (p-mTOR, Ser2448), cleaved caspase 3, pro-caspase-3, Bcl-2, Bax and glyceraldehyde-3-phosphate dehydrogenase (GADPH) were ordered from Affinity (Changzhou, China). DB was obtained from ChemFaces (Wuhan, China). Compound C (CC), the specific AMPK signaling blocker, was purchased from MedChemExpress (Shanghai, China).
7
Protein Expression Analysis of HIF-1α, Beclin-1, and LC3
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Cell culture medium was collected, cells were lysed, total protein was extracted, and protein was separated using 8% and 12% SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skim milk for 1.5 h at room temperatureat. Blots were incubated with the primary antibodies overnight, washed, incubated with secondary antibodies for 1.5 h, and washed again. Enhanced chemiluminescence (ECL) was used for color development and exposure. Bands detected using WB were scanned in grayscale mode using a multifunction imager (Thermo Fisher Scientific, Waltham, MA, USA). The protein expression of HIF-1α, Beclin-1, and LC3 was then detected. Finally, ImageJ software was used to quantify the WB bands. The antibodies used in this experiment were as follows: HIF-1α (1:1000; Affinity), Beclin-1 (1:1000; Affinity), LC3A/B (1:1000; CST), and GAPDH (1:3000; ZENBIO), Goat Anti-Rabbit IgG(H+L) HRP (1:20000; MULTISCIENCES)
8
Senescence Evaluation in Cell Cultures
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Penicillin, streptomycin and HRP-conjugated goat anti-rabbit IgG (H+L) were obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China), the senescent cell histochemical staining kit was obtained from Beyotime Institute of Biotechnology (Haimen, China), and the cell counting kit-8 (CCK-8) assay was obtained from Tongren Institute of Chemistry (Japan). VEGF and bFGF enzyme-linked immunosorbent assay (ELISA) kits were purchased from Wuhan Yunkelong Technology Co., Ltd. (SEA143Ra and SEA551Ra, Hubei, China). SIRT1 and FoxO3a were both from Cell Signaling Technology (#9475 and #2497, Danvers, MA, USA), LC3 was from Abcam (ab63817, Cambridge, MA, USA), Beclin1 was from Affinity Biosciences (AF5128, OH, USA) and p21 and p16 were obtained from Hua Biotechnology Co., Ltd., (ER1914-57 and ET1602-9, Zhejiang, China).
9
Protein Expression Analysis in Lung Cells
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According to previous protocols, 24 proteins were collected from frozen cells and lungs by a RIPA buffer, then separated with 12% SDS-PAGE. The separated proteins were transferred to PVDF membranes, and incubated with appropriate primary antibodies overnight at 4°C. The antibodies included NOX4 ( # ab154244, 1:1000, Abcam), ATG5 ( # DF6010, 1:1000, Affinity), Beclin-1 ( # AF5128, 1:1000, Affinity) and GAPDH ( # AF7021, 1:1000, Affinity). The second antibody Goat anti-Rabbit IgG (H+L) HRP ( # S001, 1:1000, Affinity) was cultured with the washed membranes for 60 min. An enhanced chemiluminescence reagent kit was used to visualize the signals.
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