Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

This protocol allows to evaluate the ubiquitylation of a specific substrate by an E3 ligase. We know that the characterization of ligase-substrate interaction is crucial to understanding their function in cells. This technique does not require expensive reagents or sophisticated equipment.

It needs plasmids and coding the genes of interest, developing an immunoprecipitation assay with cell lysates, and a Western blot to detect substrate ubiquitylation. Several human disease like cancer and neurological disorders are associated with dysregulation of E3 ligase. Hence, the identification of substrate ubiquitylation by specific E3 ligase could assist in treating or diagnosing those diseases.

Demonstrating the procedure will be by Valentine Spagnol. To begin, grow HEK293T cell line to 80 to 90%confluence in Dulbecco's modified Eagles medium supplemented with 10%fetal bovine serum and 100 units penicillin, 100 micrograms streptomycin, and 0.292 milligrams per milliliters L-glutamine. Then, incubate this culture at 37 degrees Celsius in a humidified cell culture incubator at 5%carbon dioxide.

Passage the cells by aspirating the media from the culture dish using a serological pipette and wash them once with one milliliter of sterilized 1X PBS. Next, detach the cells by adding one milliliter of trypsin ethylenediaminetetraacetic acid solution and after five minutes of incubation at 37 degrees Celsius, resuspend these cells in two milliliters of growth media using a serological pipette. Transfer the cell suspension to a fresh and clean 15-milliliter tube and centrifuge at 500 times G for five minutes at room temperature.

Then, gently remove the supernatant and resuspend the cell pellet in three milliliters of growth media by pipetting up and down to obtain a homogenous cell suspension. Then, transfer one milliliter of this cell suspension to a 100-millimeter TC-treated culture dish containing nine milliliters of growth media. Before transfection, certify if the cells are free from contamination and at an adequate confluence for transient transfections.

For each transfection sample, prepare the DNA-polyethyleneimine complexes by diluting three micrograms of each plasmid in 100 microliters of Opti-MEM 1 reduced serum medium without supplementation and mixing it gently by pipetting the solution up and down. Next, thaw the DNA-polyethyleneimine at room temperature and add it into the solution following a proportion of three microliters of DNA-polyethyleneimine per one microgram of DNA. Homogenize the solution by pipetting up and down.

Then, incubate it for 15 minutes at room temperature to allow the formation of DNA-polyethyleneimine complexes. Add the total volume of DNA-polyethyleneimine complexes to each dish containing the cell culture and mix it gently by rocking the plate back and forth. Then, incubate these cells at 37 degrees Celsius in a humidified cell culture incubator.

After the incubation and six hours prior to cell lysis, treat the transfected cells with 10-micromolar proteasome inhibitor MG132 and incubate the cells in the humidified cell culture incubator. Next, aspirate the media from each culture dish using a serological pipette and wash the cells once with one milliliter of 1X PBS. Then, detach the cells by adding one milliliter trypsin and incubate the dish at 37 degrees Celsius for five minutes.

Resuspend the cells in one milliliter of growth media and transfer the cell suspension into a fresh and clean two-milliliter microtube and centrifuge it at 500 times G for five minutes at room temperature. Once centrifugation is over, remove the supernatant by pouring it out carefully and gently resuspend the cell pellet in 200 microliters of ice-cold MP40 lysis buffer supplemented with the protease and phosphatase inhibitors cocktail. Then transfer this solution into a clean 1.5-milliliter microtube.

Incubate this cell lysate for 30 minutes on ice, and after the incubation, centrifuge the cell lysates at 16, 900 times G for 20 minutes at four degrees Celsius. Meanwhile, equilibrate the agarose anti-HA beads with ice-cold MP40 lysis buffer. Use 15 microliters of agarose anti-HA beads for each sample.

Wash the beads with 200 microliters of MP40 lysis buffer by pulsing them in a micro-centrifuge tube at 3, 000 times G for one minute at four degrees Celsius. Next, aspirate and discard the supernatant carefully with a pipette and repeat this process three times. Afterward, keep the beads equilibrated on ice until use.

After centrifuging the cell lysates, recover the supernatant and quantify the protein content in the total lysate using the Bradford method to ensure that each sample subjected to immunoprecipitation presents an equal amount of protein. Incubate the necessary volume of cell lysate with the equilibrated agarose anti-HA beads for four hours, gently rotating in a rotating incubator at four degrees Celsius, which allows the UXT-V2-HA to bind to the agarose anti-HA beads. Collect the agarose anti-HA beads by pulsing them in a microcentrifuge tube at 3, 000 times G for one minute at four degrees Celsius.

Carefully aspirate and discard the supernatant. Wash the beads three times with ice-cold MP40 cell lysis buffer and twice with ice-cold FLAG HA buffer. After the final wash, remove all the supernatant carefully using a pipette and elute the poly-ubiquitylated protein with 300 micrograms per milliliter of HA peptide diluted on FLAG HA buffer.

Incubate the agarose anti-HA beads with HA peptide for one hour at four degrees Celsius in a rocking shaker platform. Next, spin down the beads at 3, 000 times G for two minutes at four degrees Celsius and carefully remove the supernatant containing the poly-ubiquitinated proteins. If necessary, store the eluent in a fresh and clean microtube at minus 20 degree Celsius.

Resolve the eluents and the cell lysates in 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. To perform a wet transfer Western blotting, place the gel in a transfer sandwich composed of filter paper, gel membrane filter paper, cushion it with pads, and press it together by a support grid. Then, place this system vertically in a tank filled with transfer buffer in between stainless steel or platinum wire electrodes.

The transfer occurs for 90 minutes at 150 volts in a wet transfer buffer. Finally, probe the immunoblot membrane using anti-myc antibody. The specific poly-ubiquitylation of UXT-V2-HA mediated by F-box protein 7 in cells was observed after probing the eluent from anti-HA immunoprecipitation with an anti-myc antibody, which detects the myc-ub conjugated to the substrate.

The specificity of anti-myc for poly-ubiquitylated substrate was visualized in lanes one and two, wherein a smear of poly-ubiquitylated proteins was not detected when cells were co-transfected with F-box protein 7 and myc-ub in the absence of UXT-V2-HA plasmid. Additionally, no smear corresponding to protein poly-ubiquitination was visualized in the combination of F-box protein 7 and UXT-V2-HA without the myc-ub plasmid. When an empty-vector wild-type F-box protein 7 or F-box protein 7 mutant unable to assemble active SCF was transected in combination with UXT-V2-HA and myc-ub, a strong smear signal of poly-ubiquitylated UXT-V2 was observed only for wild-type F-box protein 7, suggesting that UXT-V2 was poly-ubiquitylated by SCF F-box protein 7 complex and cells compared to the controls.

The mono-precipitation steps, including incubation of cell lysates with the equilibrated beads and elution of immunoprecipitated proteins are essential to achieve the aiming of this protocol. After this procedure, in vitro ubiquitylation assay should be performed to confirm the specificity of the substrate ubiquitylation by the E3 ligase.