Experiment 3 Analysis of Sp3 Post-Translational Modification

Experiment 3 Analysis of Sp3 Post-Translational Modification

Experiment 3 Analysis of Sp3 post-translational modification

Materials

  • Tissue culture plates with brain endothelial cells (similar plates with Caco-2 cells are also available, but they are not used in this activity)
  • Verapamil (may be added to tissue culture medium up to 20 µg ml−1)
  • Mithramycin (may be added to tissue culture medium up to 10 µg ml−1)
  • Tissue culture medium
  • Rainbow markers 225 000 blue, 150 000 red, 98 000 green, 68 000 yellow, 45 000 purple, 36 000 orange, 24 000 blue, 17 000 red.
  • Detection antibodies
  • antibody to human Sp3
  • antibody to ubiquitin
  • antibody to SUMO
  • antibody to phosphotyrosine-Sp3
  • mouse antibody to human TATA-binding protein TBP.

[All supplied at an optimum dilution for detection on Western blots.]

Guidance

Devise an experiment to determine whether treatment of brain endothelial cells with either verapamil 0–20 g ml−1 or mithramycin 0–10 g ml−1 for 16 hours, causes any change in the post-translational modification of Sp3. Start with verapamil as indicated in the protocol, and then carry out a similar experiment using mithramycin.

Note that high doses of the antibiotics for extended periods may be cytotoxic, so a pilot experiment in the tissue culture laboratory, to determine a suitable range, is advisable.

[The protocol below lists all the steps needed to run this experiment. The key steps which you carry out in the onscreen laboratory are in bold; other steps are performed for you.]

Protocol

Apply the treatments to brain endothelial cells and select the incubation time.

  1. Prepare a suitable range of dilutions of verapamil in tissue culture medium.
  2. Remove medium from the brain endothelium and add the treatments.
  3. Incubate the cells for 16 hours (recommended – other incubation times can be chosen).
  4. Harvest the cells and prepare nuclear protein preparations.

Prepare samples for Western blot analysis

  1. Quantitate the protein and adjust each sample to 0.5 mg ml-1.
  2. Prepare SDS-PAGE samples by addition of 20 l reducing sample buffer to 100 µl of each sample and heat at 95C for 5 min.

Do the samples contain similar amounts of protein? Analyse the nuclear proteins by Western blot, using the antibody against TBP.

  1. Prepare an SDS polyacrylamide gel suitable to resolve TBP.
  2. Load 20 l of each sample onto a lane of the gel and 10 l rainbow markers onto an empty lane.
  3. Run the gel for 45 minutes at 100 V and then blot.
  4. Use 2 ml of primary antibody to TBP for the first stage of the immunostaining.
  5. Visualise the blot by chemiluminescence and record observations from visual inspection of the blot.

Analyse the nuclear proteins by Western blot, using the antibody against Sp3.

  1. Prepare an SDS polyacrylamide gel suitable to resolve Sp3.
  2. Load 20 l of each sample onto a lane of the gel and 10 l rainbow markers onto an empty lane.
  3. Run the gel for 45 minutes at 100 V and then blot.
  4. Use 2 ml of primary antibody to Sp3 for the first stage of the immunostaining.
  5. Visualise the blot by chemiluminescence.

Take screen shots of your blot with anti-Sp3 and incorporate it with these notes in your experimental notebook. Use your blot to determine the Mr values of the Sp3 isoforms in nuclear extracts from untreated cells.

Now determine: (i) whether any of the variants in untreated brain endothelial cells could be a result of post-translational modification; (ii) whether verapamil affects post-translational modification of Sp3 in these cells.

  1. Probe replicate blots of nuclear lysates with antibodies to phosphotyrosine, SUMO and ubiquitin.
  2. Visualise the blots and relate the findings to the results seen using an anti-Sp3 antibody, identifying any of the Sp3 bands that have undergone post-translational modification.

Carry out a similar investigation with mithramycin.

Use these data to determine:

  • Are there any data or evidence for different isoforms of Sp3?
  • Are there any data or evidence on post-translational modification of Sp3?
  • What post-translational modifications of Sp3 are present in untreated cells?
  • What effect does verapamil have on any post-translational modifications?
  • What effect does mithramycin have on any post-translational modifications?
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