Antitumor drug screening method using RhoB inhibiting ubiquitination degradation of Smurf1 as therapeutic target
An anti-tumor drug, ubiquitination technology, applied in the screening of compounds, biochemical equipment and methods, biological testing and other directions, can solve problems such as the weakening of exercise ability, and achieve the effect of clear function, wide application range and high sensitivity
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Embodiment 1
[0055] Western blotting was used to detect the effect of Smurf1 on the stability of RhoB protein.
[0056] The test method is as follows:
[0057] 1) Cell transfection:
[0058] The cells were seeded in a 12-well culture plate, transfected 24 hours later, and the cells were replaced with fresh culture medium before transfection. The transfection steps are: take a 1.5mL centrifuge tube, add 180μL of water, the corresponding plasmid, 2.5M CaCl 2 20 μL, HBS 200 μL, mix gently, let stand at room temperature for 15-30 minutes, then slowly add dropwise to the culture medium, shake the culture plate gently to make the distribution even.
[0059] After 12 hours of transfection, the cell culture medium was replaced with fresh DMEM (Dulbecco's Modified Eagle's Medium), and after continuing to culture for 12 hours, the cells were routinely collected for subsequent experiments.
[0060] Solution recipe:
[0061] HBS: 280mM NaCl, 10mM KCl, 1.5mM NaCl 2 HPO 4 , 12mM Glucose, 50mM HEPE...
Embodiment 2
[0077] The combination of Smurf1 and RhoB was detected by GST-Pulldown.
[0078] The test method is as follows:
[0079] 1) Protein expression and purification in vitro
[0080] First, clone the target DNA fragment into a suitable bacterial expression vector, transform E.coliBL-21 or DH-5α cells with the correct plasmid, select transformants on LB / ampicillin plate, and inoculate into appropriate amount of LB / amp medium , 37°C shaking culture overnight. The next day, use this as a seed, inoculate it into a large volume of LB / amp medium at a ratio of 1:100, and grow it to OD with shaking at 37°C 600 It is about 0.6, add IPTG to a final concentration of 1mmol / L to induce the expression of the fusion protein, induce at a low temperature of 20°C to reduce the formation of inclusion bodies, and continue to culture for more than 6h. For different carriers or different proteins, the optimal bacterial concentration before induction, the IPTG concentration required for induction, the...
Embodiment 3
[0087] Intracellular ubiquitination assay was used to detect whether Smurf1 affects the ubiquitination level of RhoB.
[0088] The test method is as follows:
[0089] With 1.5~2×10 5 Cells were seeded at a density of 1 / mL in a 10 cm cell culture dish, and when the cell density reached 40% to 50%, the corresponding combination of plasmids was transfected by the calcium phosphate method. 24 hours after transfection, the medium was changed and drugs were added at the same time. Cells were treated with 10 μM MG-132. The purpose of adding drugs was to inhibit the degradation of substrate proteins that had been ubiquitinated to varying degrees through the lysosomal pathway. Cells were collected 36 hours after transfection, and treated with The TNTE (0.5%) buffer of PMSF and NEM was lysed. From the obtained 1mL cell lysate, take 45μL as total lysate and add 15μL 4×loading buffer for Western blot analysis, and the remaining 955μL is used for the first IP. For the first IP, add 1 μL...
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