Application of deubiquitinating enzyme USP20 in lowering cholesterol synthesis and improving metabolic syndrome
A deubiquitinating enzyme, cholesterol technology, applied in the field of deubiquitinating enzymes, can solve problems such as ambiguity and increased cholesterol biosynthesis
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Embodiment 1
[0152] Example 1 Cell Culture
[0153] CHO-7, Huh-7 and HEK293T cells were incubated at 37°C and 5% CO 2 monolayer growth in an environment. Huh-7 and HEK293T cells were maintained in medium A (DMEM containing 100 units / mL penicillin and 100 mg / mL streptomycin sulfate), supplemented with 10% fetal bovine serum (FBS). CHO-7 and CHO-gp78-KO cells were maintained in medium B (a 1:1 mixture of DMEM and Ham's F-12 medium containing 100 units / mL penicillin and 100 mg / mL streptavidin sulfate) supplemented with 5% FBS white). Cholesterol-starved medium C was obtained from medium A or medium B supplemented with 5% lipoprotein-deficient serum (LPDS), 1 μM lovastatin and 10 μM mevalonic acid. Primary mouse hepatocyte culture medium D (M199) supplemented with 5% FBS, 100 units / mL penicillin and 100 μg / mL streptomycin sulfate.
Embodiment 2
[0154] Example 2 Western blotting
[0155] The harvested cells or tissues are lysed in RIPA lysis buffer supplemented with protease inhibitors. RIPA lysis buffer contained 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM MgCl2, 1.5% NP-40, 0.1% SDS and 0.5% sodium deoxycholate. Protease inhibitors included MG-132 at 10 μM, leupeptin at 10 μg / ml, 1 mM phenylmethylsulfonyl fluoride, pepstatin at 5 μg / ml, N-acetylleucinal-leucinal-norleucinal at 25 μg / ml and dithiothreitol at 1 mM. The protein concentration of the lysates was determined using the BCA method (Thermo Fisher Scientific). Protein samples were mixed with membrane lysis buffer (62.5mM Tris-HCl, pH 6.8, 15% SDS, 8M urea, 10% glycerol and 100mM DTT) and 4x loading buffer (150mM Tris-HCl, pH 6.8, 12% SDS, 30% glycerol, 6% 2-mercaptoethanol and 0.02% bromophenol blue) were incubated at 37°C for 30 minutes. Protein samples were separated by SDS-PAGE gel and then transferred to PVDF membrane. Block with TBS containing 0.075% Tw...
Embodiment 3
[0157] Example 3 Immunoprecipitation
[0158] Cells were washed twice with ice-cold PBS buffer, and then harvested in 600 μl of ice-cold lysis buffer (PBS containing 0.5% digitonin, 5mM EGTA, 5mM EDTA, protease and phosphatase inhibitors). After the whole cell lysate was centrifuged (12,000g, 10 minutes), the precipitate was discarded, and 60 μl of the supernatant was used as the whole cell lysate control (Input component), and 500 μl of the supernatant was mixed with 30 μl of the primary antibody. Rotate and incubate for 2 hours, centrifuge at 1000g for 5 minutes, take 60 μl of supernatant as the supernatant control (Supernatant component), then discard the remaining supernatant, wash 3 times with lysis buffer, each time for 10 minutes and keep rotating to mix . Finally, the precipitate was collected, and 120 μl of SDS PAGE Loading buffer was added, marked as the pellet fraction. Then each component was detected by Western blot.
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