HEK cells were transfected with the indicated amount of PAK1-DN or PAK1-CA plasmids, incubated for 36 h, treated with 10 μM lactacystin (Tocris) for 12 h and subjected to immunoprecipitation with anti-Bcl-2 antibody (Cell Signaling). Immunoprecipitates were subjected to Western blot analysis using anti-monoclonal mono- and polyubiquitinylated conjugates antibody (FK2, BML-PW8810, Enzo Life Sciences, Farmingdale, NY, USA).
Lactacystin
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
Lactacystin is a laboratory reagent that functions as a highly specific and potent inhibitor of the 20S proteasome, a multi-subunit proteolytic complex found in the cytoplasm and nucleus of eukaryotic cells. It blocks the proteolytic activity of the proteasome, thereby altering the degradation of cellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl 2 antibody, by Cell Signaling Technology (1 mentions)
Rapamycin, by Thermo Fisher Scientific (1 mentions)
Betulinic acid, by Bio-Techne (1 mentions)
Cd 1 mice, by Charles River Laboratories (1 mentions)
Mg132, by Bio-Techne (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
4 protocols using lactacystin
1
Investigating Bcl-2 Ubiquitination in HEK Cells
2
Modulation of Autophagy and Proteasome Pathways
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24 hours after transfection, Hepa1-6 cells were divided into 12-well plates. After 24–48 hours, cells were subject to drug treatments for indicated lengths of time.
The following autophagy inhibitors and agonists were used: autophagy inhibitor 3-methyladenine, (3MA) stock solution: 100mM in water (Sigma Cat#: M9281) was used at 5mM final concentration; autophagy inhibitor wortmannin, stock solution: 0.1mM in DMSO, (Acros Organics, Cat#: 32859) was used at 200nM final concentration; autophagy agonist rapamycin, stock solution: 1mM in DMSO (Fisher Scientific, Cat#: BP29631) was used at 5μM final concentration; autophagy agonist lithium, stock solution: 1M in water, (Fisher Scientific, Cat#: L121-100) was used at 10mM final concentration.
The following proteasome inhibitors were used: lactacystin, stock solution: 10mM in DMSO (Tocris Bioscience, Bristol UK, Cat#: 2267) was used at 10uM final concentration; MG-132 stock solution:10mM in DMSO (Boston Biochem, Cat#: I-130) was used at 5μM final concentration.
For cycloheximide chase, cycloheximide was used at 25μg/ml (Stock solution: 2.5mg/ml in water, Acros Organics, Cat#:357420050). Cells were harvested at indicated time points.
The following autophagy inhibitors and agonists were used: autophagy inhibitor 3-methyladenine, (3MA) stock solution: 100mM in water (Sigma Cat#: M9281) was used at 5mM final concentration; autophagy inhibitor wortmannin, stock solution: 0.1mM in DMSO, (Acros Organics, Cat#: 32859) was used at 200nM final concentration; autophagy agonist rapamycin, stock solution: 1mM in DMSO (Fisher Scientific, Cat#: BP29631) was used at 5μM final concentration; autophagy agonist lithium, stock solution: 1M in water, (Fisher Scientific, Cat#: L121-100) was used at 10mM final concentration.
The following proteasome inhibitors were used: lactacystin, stock solution: 10mM in DMSO (Tocris Bioscience, Bristol UK, Cat#: 2267) was used at 10uM final concentration; MG-132 stock solution:10mM in DMSO (Boston Biochem, Cat#: I-130) was used at 5μM final concentration.
For cycloheximide chase, cycloheximide was used at 25μg/ml (Stock solution: 2.5mg/ml in water, Acros Organics, Cat#:357420050). Cells were harvested at indicated time points.
3
Morphine and Lactacystin Administration Protocol
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Morphine hydrochloride (morphine, Mor) was purchased from Qinghai Pharmaceutical Manufactory (China). Lactacystin (LAC) was purchased from Tocris. Morphine was dissolved in 0.9% saline and administered subcutaneously (s.c.) at a volume of 0.1 mL/100 g bodyweight. LAC was dissolved in 72.5% dimethyl sulfoxide. Drug doses were adopted as follows: morphine (1, 3, or 10 mg/kg, s.c.) and LAC (400 or 800 ng/rat, microinjection). All the drugs were freshly prepared before the experiments.
4
Isolation and Treatment of Cortical Neurons
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Timed pregnant CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Cerebral cortical neurons were prepared as described previously [21 (link)]. Briefly, cerebral cortices from embryos at E16 were aseptically dissected and plated at 3.5 × 105 cells per well on polyethyleneimine-coated 6-well plates. Neurons were cultured in Neurobasal medium supplemented with 2 mM L-glutamine, B27 and antibiotics (Invitrogen). The media was replaced with Neurobasal supplemented with B27 minus antioxidants, glutamine, and antibiotics on the third day in vitro (DIV 3). Treatment of neurons was conducted at DIV 14. The following pharmacological treatments were used: MG132 (20 μM); lactacystin (50 μM); or betulinic acid (20 μg/mL) (all the reagents were from Tocris Biosciences, Minneapolis, MN, USA). At the end of the treatments, cells were washed in Phosphate Buffered Saline (PBS) and lysed in ice-cold lysis buffer supplemented with protease inhibitor cocktail (Sigma) for immunoblotting.
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