Bafilomycin a1 baf a1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Bafilomycin A1 (Baf A1) is a specific inhibitor of vacuolar-type H+-ATPase (V-ATPase), a proton pump that acidifies various cellular compartments. It is commonly used as a tool compound in cell biology research to study the role of V-ATPase-mediated acidification in various cellular processes.

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Lab products found in correlation

Jnk1 2, by Cell Signaling Technology (3 mentions) P jnk1 2, by Cell Signaling Technology (3 mentions) Phosphatase inhibitor cocktail, by Merck Group (3 mentions) P erk1 2, by Cell Signaling Technology (3 mentions) P akt, by Cell Signaling Technology (3 mentions) Erk1 2, by Cell Signaling Technology (3 mentions) β actin, by Cell Signaling Technology (3 mentions) Protease inhibitor cocktail, by Merck Group (3 mentions) Torin1, by Bio-Techne (2 mentions) Dapi dye, by Merck Group (2 mentions) Z vad fmk, by Santa Cruz Biotechnology (2 mentions) Rnase a, by Merck Group (2 mentions) Trehalose, by Merck Group (2 mentions) Beclin 1, by Cell Signaling Technology (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Propidium iodide, by Merck Group (2 mentions) 3 methyladenine 3 ma, by Merck Group (2 mentions) Sodium metasilicate solution, by Merck Group (1 mentions) Lamp2 ab13524, by Abcam (1 mentions)

9 protocols using bafilomycin a1 baf a1

Chloroquine Inhibition of Autophagy

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Chloroquine (CQ) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Bafilomycin A1 (Baf A1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Shanghai, China). LC3 (#3868), HDAC6 (#7612), acetyl-α-tubulin (#5335), and α-tubulin (#2125) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). SQSTM1/p62 (#ab56416), LAMP1 (#ab24170), and LAMP2 (#ab13524) primary antibodies were purchased from Abcam (Cambridge, UK). GAPDH (#60004-1-lg), VAMP8 (#15546-1-AP), SNAP29 (#12704-1-AP), and STX17 (#17815-1-AP) primary antibodies were purchased from Proteintech Group (Boston, MA, USA). The second antibodies (peroxidase-conjugated goat anti-rabbit IgG and peroxidase-conjugated goat anti-mouse IgG) were purchased from Yeasen Biotechnology (Shanghai, China). The sodium metasilicate solution was prepared as previously reported at a stock solution of high concentration (100 mM) with some modification [23 (link), 24 (link)]. In brief, sodium metasilicate solution (Sigma-Aldrich, #338443) was diluted into alpha-MEM to prepare a stock solution. The pH was adjusted using HCl to a physiological range (pH about 7.4) before 10% fetal bovine serum (FBS; ScienCell, Carlsbad, CA, USA) was added.

Li Z., Liu S., Fu T., Peng Y, & Zhang J. (2019). Microtubule destabilization caused by silicate via HDAC6 activation contributes to autophagic dysfunction in bone mesenchymal stem cells. Stem Cell Research & Therapy, 10, 351.

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Immunostaining Protocol for Protein Analysis

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Reagents used in this study include bafilomycin A₁ (BafA₁, Santa Cruz Biotechnology, sc-201550), DAPI (Invitrogen, P36931), Hoechst 33342 (Invitrogen, H3570), IL-4 (Peprotech, 214-14), LPS (Sigma, L2654), Phalloidin Alexa Fluor™ 594 (Invitrogen, A12381), ProLong Gold antifade mountant with DAPI (Life technologies, P36935) and Torin1 (Tocris, 4247). Primary antibodies were used according to manufacturer’s instructions. A list of primary and secondary antibodies used in this study is shown in Table 1.Table 1

List of antibodies

Antibodies	Companies
Primary antibodies
ACTB, mouse monoclonal antibody	Sigma-Aldrich (A-3853)
APOE, rabbit monoclonal antibody	Abcam (ab183597)
ARG1, rabbit polyclonal antibody	Abcam (ab91279)
ATG7, rabbit polyclonal antibody	Abcam (ab133528)
GAPDH, mouse monoclonal antibody	Millipore (CB1001)
LAMIN A/C, mouse monoclonal antibody	Sigma-Aldrich (SAB4200236)
LC3B, rabbit polyclonal antibody	Sigma-Aldrich (L7543)
NOS2, rabbit polyclonal antibody	Cell signaling (13120)
P65, NF-κB subunit, rabbit polyclonal antibody	Santa Cruz (sc-372)
Secondary antibodies
IRDye^® 800CW Goat anti-Mouse IgG	Li-COR (926-32210)
IRDye^® 680RD Goat anti-Rabbit IgG	Li-COR (926-68071)
Donkey anti-Rabbit IgG, Alexa Fluor 488	Invitrogen (A21206)

List of primary and secondary antibodies used in this study, the respective companies and the catalogue numbers

Friess L., Cheray M., Keane L., Grabert K, & Joseph B. (2021). Atg7 deficiency in microglia drives an altered transcriptomic profile associated with an impaired neuroinflammatory response. Molecular Brain, 14, 87.

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Investigating Cellular Responses to Oxidative Stress

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Ammonium tetrathiomolybdate (TTM, #323446) and chloroquine diphosphate salt (CQ, #C6628) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MG132 (#S1748) and dihydroethidium (DHE, #S0063) were obtained from Beyotime (Shanghai, China). Tert-butylhydroquinone (tBHQ, #HY-100489) was purchased from MedChemExpress (Shanghai, China). Bafilomycin A1 (BafA1, #sc-201550) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Sodium arsenite (NaAsO₂, #H4525) was obtained from Xiya Reagent (Shandong, China). 7-aminoactinomycin D (7-AAD, #AP104) and Annexin V FITC/ Propidium iodide (PI) apoptosis kit (#70-APCC101-100) were obtained from MultiSciences (Hangzhou, China). Dulbecco’s Modified Eagle Medium (DMEM, #C11995500BT) was purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS, #S711-001S) was purchased from Lonsera (Shanghai, China). Penicillin-streptomycin (#15140122) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Puromycin (#P8230) was purchased from Solarbio (Beijing, China). CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) kit (#G3581) was obtained from Promega (Madison, WI, USA).

Zhang M., Qiu H., Mao L., Wang B., Li N., Fan Y., Weng P., Hu S., Dong X., Qin X., Chen C., Zou Z., Yu C, & Zhang J. (2022). Ammonium tetrathiomolybdate triggers autophagy-dependent NRF2 activation in vascular endothelial cells. Cell Death & Disease, 13(8), 733.

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Coronarin D Induces Apoptosis via Oxidative Stress

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Coronarin D (purity > 95%) was purchased from ChemFaces (Wuhan, Hubei, China). It was dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium to the final concentration on the experimental day. The final concentration of DMSO for all treatments was consistently less than 0.1%. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), RNase A, DAPI dye, protease inhibitor cocktail, phosphatase inhibitor cocktail, monodansylcadaverine (MDC), acridine orange (AO), and 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibody against cleaved caspase-3, -8, and -9, cleaved poly (ADP-ribose) polymerase (PARP), LC3B, p62, NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2 TSC1, TSC2, Rheb, p-mTOR (Ser2448), mTOR, p-AKT, AKT, p-ERK1/2, ERK1/2, p-p38, p38, p-JNK1/2, JNK1/2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Specific inhibitors for N-Acetyl-L-cysteine (NAC) AKT inhibitor (LY294002), ERK1/2 (U0126), p38 MAPK (SB203580), JNK (SP600125) Wortmannin and Bafilomycin A1 (Baf A1), and z-VAD-FMK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Chen J.C., Hsieh M.C., Lin S.H., Lin C.C., Hsi Y.T., Lo Y.S., Chuang Y.C., Hsieh M.J, & Chen M.K. (2017). Coronarin D induces reactive oxygen species-mediated cell death in human nasopharyngeal cancer cells through inhibition of p38 MAPK and activation of JNK. Oncotarget, 8(64), 108006-108019.

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Molecular Mechanism of Cell Death Regulation

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Reagents used in this study include bafilomycin A₁ (BafA1) (Santa Cruz Biotechnology, sc-201,550), carbamazepine (CBZ) (Sigma-Aldrich, C4024), clonidine (Sigma-Aldrich, C7898), Hoechst 33,342 (Molecular probes/Invitrogen, H3570), rapamycin (LC Laboratories, R5000), torin1 (Tocris, 4247), trehalose (Sigma-Aldrich, T9531), staurosporine (STS) (Sigma-Aldrich, S6942), 5-aza-2´-deoxycytidine (5-AZA-CdR) (Sigma-Aldrich, A3656) and ProLong^TM Gold antifade Mounting medium with DAPI (Molecular probes/Invitrogen, P36931).
Primary antibodies used in this study include ACTB (mouse monoclonal antibody [mAb]; Sigma Aldrich, A-3853), ATG5 (mouse mAb; Bio-techne, 603,813), ATG7 (rabbit polyclonal antibody [pAb]; Genetex, GTX61647), CASP3 (rabbit mAb; Cell Signaling Technology, 9662), DNMT1 (mouse mAb; Abcam, ab13537), DNMT3A (rabbit pAb; Abcam, ab2850 and Santa Cruz Biotech., sc20703), DNMT3B (mouse mAb; Abcam, ab13604), GAPDH (rabbit pAb; Trevigen, 2275), H3 C-terminal (rabbit pAb; Active Motif, 39,164), LMNB1 (rabbit pAb; Abcam, ab16048), LC3B (rabbit pAb; Sigma Life Science, L7543), PARP1 (rabbit mAb; Cell Signaling Technology, 9532), and SQSTM1/p62 (rabbit pAb; Cell Signaling Technology, 5114).

González-Rodríguez P., Cheray M., Füllgrabe J., Salli M., Engskog-Vlachos P., Keane L., Cunha V., Lupa A., Li W., Ma Q., Dreij K., Rosenfeld M.G, & Joseph B. (2020). The DNA methyltransferase DNMT3A contributes to autophagy long-term memory. Autophagy, 17(5), 1259-1277.

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Hesperetin Modulates Autophagy Pathways

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Hesperetin (Cat. No. 520-33-2) was purchased from ChemFaces Company (ChemFaces, Wuhan, China) and a 100 mM stock solution was prepared in DMSO (0.1% v/v final concentration) and stored at −20 °C. 3-Methyladenine (3-MA, an autophagy inhibitor) was purchased from Sigma Chemical Co. (St. Louis, MO, USA), and bafilomycin A1 (Baf-A1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti-p-AMPKα (Thr172; #2535), polyclonal anti-AMPKα (D63G4) (#5832), polyclonal anti-p-mTOR (Ser2448; #5536), polyclonal anti-mTOR (7C10) (#2983), polyclonal anti-cleaved PARP (#9532), polyclonal anti-Beclin-1 (#3495), polyclonal anti-SQSTM1/p62 (#5114), polyclonal anti-p-Akt (Ser4723; #4060), polyclonal anti-p-Akt (#9272), polyclonal anti-Atg5 (#2630), monoclonal anti-Bcl-2 (#15071), monoclonal anti-Bax (#5023), and monoclonal anti-GAPDH (#0411) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). We also purchased polyclonal-anti-LC-3B (#L7543; Sigma, UK), goat anti-rabbit IgG secondary antibodies (#NA934; GE Healthcare Life Sciences, Chalfont, UK), and IgG HRP-conjugated secondary antibody (polyclonal anti-mouse; #115-035; 1:5000; Jackson ImmunoResearch Laboratories, UK).

Lin C.Y., Chen Y.H, & Huang Y.C. (2023). Hesperetin Induces Autophagy and Delayed Apoptosis by Modulating the AMPK/Akt/mTOR Pathway in Human Leukemia Cells In Vitro. Current Issues in Molecular Biology, 45(2), 1587-1600.

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Evaluation of Chemotherapeutic Agents on Cell Viability

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For some experiments, cells were treated with doxorubicin (Dx, 1 μM, Sigma-Aldrich), etoposide (Eto, 10 μM, Sigma-Aldrich), 5-fluorouracil (5-FU, 10 μM, Sigma-Aldrich), oxaliplatin (Oxa, 10 μM, European Pharmacopoeia Reference Standards, Strasbourg, France), cyclophosphamide (CP, 10 mM, Sigma-Aldrich), MG132 (20 μM, Santa Cruz Biotechnology, Dallas, TX, USA), trehalose (100 mM, Sigma-Aldrich), concanamycin A (Con A, 50 nM, Sigma-Aldrich), bafilomycin A1 (Baf A1, 200 nM, Santa Cruz Biotechnology), cycloheximide (CHX, 50 μg/ml, Sigma-Aldrich), ammonium chloride (40 mM, Sigma-Aldrich) or 3-methyladenine (3-MA, 10 mM, Merck, Darmstadt, Germany) at the indicated times.

Galindo-Moreno M., Giráldez S., Sáez C., Japón M.Á., Tortolero M, & Romero F. (2017). Both p62/SQSTM1-HDAC6-dependent autophagy and the aggresome pathway mediate CDK1 degradation in human breast cancer. Scientific Reports, 7, 10078.

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Melatonin Modulation of Cancer Cell Signaling

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Melatonin (purity >99%) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). It was dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium to the aimed concentration on experimental day. The final concentration of DMSO for all treatments was consistently less than 0.1%. Cell culture reagents were obtained from Invitrogen (Carlsbad, CA, USA). The VCR, Coomassie brilliant blue, MTT, 4′,6-diamidino-2-phenylindole (DAPI) dye, protease inhibitor cocktail, phosphatase inhibitor cocktail, and AO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Negative inhibitor (miRNA inhibitor negative control), miRNA-34b-5p inhibitor, and miRNA-892a inhibitor were purchased from Clontech (CA, USA). Antibody against cleaved PARP, cleaved caspase-3, -9, LC3, SQSTM1, Beclin-1, ABCB1, ABCG2, p-AKT, AKT, p-ERK1/2, ERK1/2, p-p38, p38, p-JNK1/2, JNK1/2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against ABCB4 was obtained from MyBioSource (San Diego, CA, USA). Specific inhibitors for AKT inhibitor (LY294002), ERK1/2 (U0126), p38 MAPK (SB203580), JNK (SP600125), wortmannin, bafilomycin A1 (Baf A1), and z-VAD-FMK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Hsieh M.J., Lin C.W., Su S.C., Reiter R.J., Chen A.W., Chen M.K, & Yang S.F. (2020). Effects of miR-34b/miR-892a Upregulation and Inhibition of ABCB1/ABCB4 on Melatonin-Induced Apoptosis in VCR-Resistant Oral Cancer Cells. Molecular Therapy. Nucleic Acids, 19, 877-889.

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Polyphyllin G Induces Apoptosis

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Polyphyllin G (purity > 98%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and was dissolved in dimethyl sulfoxide (DMSO) and diluted in cell culture medium to the final concentration before use. The final concentration of DMSO for all treatments was consistently less than 0.1%. All cell culture-related reagents were purchased from Invitrogen (Carlsbad, CA). DAPI dye, propidium iodide (PI), RNase A, protease inhibitor cocktail, phosphatase inhibitor cocktail, and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO). Antibody against cleaved caspase-3, -8, and -9, cleaved poly (ADP-ribose) polymerase (PARP), Bcl-2, Bcl-xL, Bax, LC3B, Beclin1, p62, p-AKT, AKT, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-p38, p38, PI3K, mTOR (ser2448), mTOR, Raptor, Rictor, GβL and β-actin were purchased from Cell Signaling Technology (Danvers, MA). Specific inhibitors for AKT inhibitor (LY294002), ERK1/2 (U0126), p38 MAPK (SB203580), JNK (SP600125), z-VAD-FMK, Wortmannin and Bafilomycin A1 (Baf A1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Chen J.C., Hsieh M.J., Chen C.J., Lin J.T., Lo Y.S., Chuang Y.C., Chien S.Y, & Chen M.K. (2016). Polyphyllin G induce apoptosis and autophagy in human nasopharyngeal cancer cells by modulation of AKT and mitogen-activated protein kinase pathways in vitro and in vivo. Oncotarget, 7(43), 70276-70289.

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