Protein half-life was determined by using a 100 μg/mL cycloheximide (CHX) for 0, 4, 8, 12, 16 or 24 h. Proteasome inhibitor MG-132 was applied for 24 h with the concentration of 0, 10, 15 or 20 μM. In time-course experiments, 15 μM MG-132 was applied for 0, 3, 6, 12 or 24 h. 300 nM Bafilomycin A1 (Baf-A1) was applied for 4 h. Baf-A1 was purchased from Abcam and other drugs were purchased from Sigma-Aldrich. The details were described in previous studies (Settembre et al., 2011 (link); Liu et al., 2013 (link); Wu and Song, 2013 (link); Medina et al., 2015 (link)).
Bafilomycin a1 baf a1
Manufactured by Abcam
Sourced in United Kingdom
Bafilomycin A1 (Baf-A1) is a macrolide antibiotic that functions as a specific and potent inhibitor of vacuolar-type H+-ATPase (V-ATPase). V-ATPase is an enzyme responsible for acidifying various intracellular compartments, such as endosomes, lysosomes, and secretory vesicles. By inhibiting V-ATPase activity, Baf-A1 disrupts the acidic environment within these organelles, leading to various cellular effects.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Baf a1, by Abcam (1 mentions)
Rapamycin, by Apexbio (1 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
3 methyladenine 3 ma, by MP Biomedicals (1 mentions)
Protein a g plus agarose beads, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
Azd6765, by Merck Group (1 mentions)
Cp 101 606, by Merck Group (1 mentions)
Dmem f12, by Welgene (1 mentions)
L glutamic acid, by Merck Group (1 mentions)
4 protocols using bafilomycin a1 baf a1
1
Protein Turnover Kinetics Determination
2
HUVEC Cell Culture and Pharmacological Modulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVECs were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin–streptomycin at 37 °C and 5% CO2 in a humidified incubator. Rapamycin (APExBIO Technology, Houston, TX, USA) was dissolved in dimethyl sulfoxide (DMSO) at 5 mM for storage at − 20 °C. The autophagy inhibitor 3-methyladenine (3-MA; MP Biomedicals, Santa Ana, CA, USA) was dissolved in ddH2O at a concentration of 200 mM for storage at room temperature, and bafilomycin A1 (BafA1, Abcam, Cambridge, UK) was dissolved in DMSO at a concentration of 400 mM and kept at − 20 °C. The ER stress antagonist 4-phenylbutyric acid (4-PBA; Yuanye Bio-Technology, Shanghai, China) was dissolved in phosphate-buffered saline (PBS) at a concentration of 500 mM for storage at − 20 °C. The final concentrations of the drugs and duration of treatments are indicated in the figure legends.
3
Characterizing Imatinib-resistant Leukemia Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
K562 and K562G cells were provided by Professor Jie Jin (The First Affiliated Hospital of Zhejiang University). The cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO2 atmosphere. Imatinib-resistant K562G cells was established by culturing cells in gradually increasing concentrations of imatinib and gained resistance to imatinib over several months of culture (Appendix I). However, K562G cells do not contain mutations within the TK domain. Autophagic inhibitor Bafilomycin A1 (Baf-A1) was purchased from Abcam, autophagic inhibitor chloroquine (CQ), proteasome inhibitor MG132 and cycloheximide (CHX) were purchased from Sigma-Aldrich (Merck KGaA). HHT was purchased from Selleckchem (cat. no. s9015). Protein A/G plus agarose beads were obtained from Thermo Fisher Scientific, Inc. (cat. no. 20423). Compounds were dissolved in dimethyl sulfoxide (DMSO) and added to culture media until it reaches a final concentration of 0.1% DMSO. For the vehicle control, 0.1% DMSO alone was used.
4
ARPE-19 Cell Culturing and Compound Treatments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human RPE cell line, ARPE-19, was cultured in Dulbecco’s modified Eagle medium, F-12 (DMEM F-12; WELGENE, Gyeongsan, Korea) containing 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2. Ro 25-6981 maleate salt, L-glutamic acid, AZD6765, and CP-101,606 were purchased from Sigma-Aldrich (St. Louis, MI, USA). A2E was purchased from Key Synthesis LLC (Philadelphia, PA, USA). Rapamycin was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Bafilomycin A1 (Baf A1) was purchased from Abcam (Cambridge, UK).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!