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10 protocols using chloroquine cq

1

Regulation of HBX Protein Stability

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HepG2.215 cell lines were seeded in 6-well plates at a density of 3 × 105 cells/well and transfected with Flag-HBX+empty vector plasmids or Flag-HBX+Myc-NEDD4 plasmids for 72 h. Protein lysates were prepared at the indicated time points after the addition of cycloheximide (CHX) (10 μM). Equal amounts of protein were separated by SDS-PAGE. The levels of Flag-HBX were determined by immunoblotting and quantified at the indicated time points. HepG2.215 cells were seeded in 6-well plates at a density of 3 × 105 cells/well and transfected with Flag-HBX and Myc-NEDD4 plasmids for 72 h. Cells were treated with chloroquine (CQ, 50 µM, Cell Signaling Technology, 14774) or MG132 (20 µM, Selleck, S2619) for another 12 h to block the autophagy-lysosome or ubiquitin-proteasome pathway, respectively. Protein lysates were harvested after that, and the protein level of Flag-HBX was evaluated by western blotting.
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2

Preparing Withaferin A and Chloroquine

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Withaferin A was purchased from ChromaDex (Irvine, CA, USA). It was dissolved in dimethylsulfoxide and stored at −20°C. Chloroquine (CQ) was obtained from Cell Signaling Technology (Danvers, MA, USA). It was dissolved in distilled water and stored at 4°C.
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3

Photosensitizer-based Targeted Therapy

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The photosensitizer meso-tetraphenyl chlorine disulfonate (TPCS2a /Fimaporfin) was provided by PCI Biotech (Oslo, Norway). TPCS2a was diluted in 3% polysorbate 80, 2.8% mannitol and 50 nM Tris (pH 8.5) to a final concentration of 0.35 mg/ml and kept at 4 °C protected from light. All work with TPCS2a was performed under subdued light. The immunotoxin CD105-saporin (Beta-015) conjugate (CD105-SAP) was a gift from Advanced Targeting Systems. CD105-SAP is a chemical 270 kDa-conjugate between a mouse monoclonal antibody (clone 43A3) to human CD105 and the ribosome-inactivating protein saporin. Lysotracker® Green (pH 5.2) (L7526, Invitrogen) was used as a marker of acidic vesicles in the live microscopy studies. Chloroquine (CQ) (#14774) was purchased from Cell Signaling, and BSO (DL-Buthionine-(S,R)-sulfoximine, B2640) was purchased from Sigma.
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4

Regulation of Smurf2 Protein Stability

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293T cells were seeded in 6-well plates at a density of 3 × 105 cells/well and transfected with Flag-Smurf2 and Myc-TRAF4 plasmids for 36 h. Cells were treated with chloroquine (CQ, 50 µM, Cell Signaling Technology, 14774) or MG132 (10 µM, Selleck, S2619) for another 12 h to block the autophagy–lysosome or ubiquitin–proteasome pathway. Protein lysates were harvested after that, and the protein level of Flag-Smurf2 was evaluated by western blot.
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5

Autophagy Regulation in Bacterial Infection

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BLP (Pam3Cys-Ser-Lys4, ab142085) was purchased from Abcam (Cambridge, UK). Gram-negative S. typhimurium (CMCC50097) and Gram-positive S. aureus (ATCC6538) were obtained from the Laboratory of Pathogenic Microorganism, Southern Medical University, Guangzhou, China. Antibodies against autophagy-related proteins (Autophagy Antibody Sampler kit #4445) and chloroquine (CQ) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl2 (#26593-1-AP), Bax (#50599-2-Ig), and Caspase3 (#19677-1-AP) were obtained from ProteinTech (Chicago, IL, USA). We purchased 3-methyladenine (3-MA) (#189490) from Sigma-Aldrich (St. Louis, MO, USA). The siRNA targeting Atg3, Atg7, and scrRNA were obtained from GenePharma (Shanghai, China).
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6

Cytotoxicity assay for tigecycline and chloroquine

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Cell viability assay was performed using Cell Counting Kit-8 (CCK8; CK40; Dojingdo, Kumamoto, Japan). Cells were seeded in 96-well plates and then exposed to tigecycline (Hisun Pharmaceutical Company, Fuyang, China) and/or chloroquine (CQ) (14774; Cell Signaling Technology, Danvers, MA) at the indicated concentrations for 48 h. The cells were incubated with CCK-8 solution for 2 h, after which absorbance was measured at 450 nm using a SpectraMax Plus microplate reader (Molecular Devices, Sunnyvale, CA, USA).
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7

Quantifying Mitophagy in Rat Hepatocytes

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Mitophagic flow was assessed by flow cytometry using a modification of a previously published protocol35 (link). RALA255-10G rat hepatocytes were cultured for 6 h with or without palmitate (0.2 mM), GDNF (100 ng/mL), and with or without the lysosomal inhibitor chloroquine (CQ) (30 µM) (Cell Signaling Technology, Danvers, MA, USA). MitoTracker Red CMX-ROS (#M7512, Molecular Probes, Eugene, OR, USA) was then added to the cells at a final concentration of 25 nM in culture medium and the cells cultured for a further 15 min. The cells were washed once in PBS, stained for 30 min with LIVE/DEAD Fixable Near-IR Stain (#L34976, Life Technologies Corp., Carlsbad, CA, USA) and fixed with 3.7% formaldehyde in complete culture medium at 37 °C for 15 min. After washing 3 times with PBS with 1% bovine serum albumin, the cells were analyzed by flow cytometry at the Emory Pediatric and Winship Flow Cytometry Core (Emory University, Atlanta, GA, USA). Mitophagic flow was calculated from the difference in the number of MitoTracker Red CMX-ROS-positive hepatocytes between cells cultured in the presence of the inhibitor (CQ) and those cultured without CQ for each treatment.
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8

Pharmacological Modulation of PTC Cells

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TPC-1 and K1 cells (human PTC cell lines) were obtained from the University of Colorado Cancer Center Cell Bank. All cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 atmosphere. All experiments were performed with mycoplasma-free cells.
The ROS scavenger N-acetyl-L-cysteine (NAC), histone deacetylase (HDAC) inhibitor trichostatin A (TSA), AMPK pathway activator AICAR, autophagy inhibitor chloroquine (CQ), ER stress inhibitor 4-phenylbutyrate (4-PBA), and proteasome inhibitor MG-132 were all purchased from Cell Signaling Technology. The working concentration and time were confirmed by the manufacturer's instructions combined with the experimental requirements.
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9

Extracellular Vesicle Isolation Protocols

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For EVs isolation from condition media, cells were cultivated for 24 h in medium supplemented with exosome-depleted serum (System Biosciences, Palo Alto, CA, USA). For experimental purposes, cells were treated with different concentrations of amiodarone (AM, Sigma-Aldrich), palmitic acid (PA, Sigma-Aldrich), chloroquine (CQ, Cell Signaling Technology, Danvers, MA, USA), bafilomycin A1 (BafA1, Cell Signaling Technology) or vehicle (0.05% DMSO, CTRL) for 24 h in the same conditions. Medium was collected and underwent serial centrifugation steps to remove cells and cell debris (300× g, 10 min and 2000× g, 10 min, respectively). L/m EVs were recovered by centrifugation at 10,000× g for 30 min (10K fraction), and the pellet was washed with PBS and then centrifuged again at 10,000× g, resuspended in PBS and stored at −80 °C. For sEVs recovery, the supernatant obtained upon 10,000 g centrifugation was ultracentrifuged at 100,000× g for 70 min (100K fraction), the resulting pellet washed with PBS, resuspended in PBS and stored at −80 °C. All PBS was 0.22 µm filtered. Protein content was determined by the Bradford method, using bovine serum albumin as standard.
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10

Modulation of PTC cell lines

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Human PTC cell lines TPC-1 and K1 were purchased from the University of Colorado Cancer Center Cell Bank. All cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) at 37 °C in a 5% CO 2 atmosphere. ROS scavenger N-acetyl-L-cysteine (NAC), histone deacetylase HDAC inhibitor Trichostatin A (TSA), AMPK pathway activator AICAR, autophagy inhibitor chloroquine (CQ), ER-stress inhibitor 4-phenylbutyrate (4-PBA), proteasome inhibitor MG-132 were all purchased from Cell Signaling Technology. The work concentration and time were con rmed by instructions combined with experimental requirements.
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