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Lab. Meeting (1 st August)

Lab. Meeting (1 st August). In Vitro studies to define the role of PERK in insulin synthesis * Using the 832/13 cell line. Experimental Design. Equal numbers of 832/13 cells were plated on dishes These were starved for the sulphur containing amino acids

carlos-barlow
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Lab. Meeting (1 st August)

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  1. Lab. Meeting(1st August) • In Vitro studies to define the role of PERK in insulin synthesis * Using the 832/13 cell line

  2. Experimental Design • Equal numbers of 832/13 cells were plated on dishes • These were starved for the sulphur containing amino acids methionine and cysteine for half an hour • Methionine and cysteine labeled with S35 was added to this medium (should be incorporated in any new proteins synthesized) • The cell lysates were harvested at different time points (This would be indicative of radiolabeling of proteins in the intracellular compartment)

  3. Determination of time point of maximum S35 incorporation • Comparison of beta emission generated signal from • Total cell lysates • (TCA precipitated) Total protein (from the cell lysates) : Gives data regarding percentage incorporation

  4. Determination of time point of maximum S35 incorporation

  5. 832/13 cells 832/13 cells with lacZ 832/13 cells with ▲C INCUBATED for 36 hours, no significant cell death at the time of harvesting = Untreated controls = (adenovirus vector without the dominant negative construct) =(adenovirus vector with the dominant negative construct) Studying protein synthesis (intracellular compartment) using S35 labeling

  6. S35 incorporation following 30 mins of pulsing Untreated lacZ ▲C

  7. Labeling as a fraction of total Protein content in samples Untreated LacZ ▲C

  8. Autoradiograph for a western of total protein (TCA precipitated samples) to check for signal

  9. Immunoprecipitation for insulin Untreated LacZ ▲C Scintillation counter readings

  10. Immunoprecipitation for insulin Untreated LacZ ▲C

  11. Immunoprecipitation for insulinNon Reducing gel (without Urea) Untreated LacZ ▲C Non Specific Aggregates?

  12. Immunoprecipitation for insulin Reducing gel (with Urea)

  13. Immunoprecipitation for insulin Untreated LacZ ▲C Suggests higher insulin synthesis in delta C treated cells

  14. Insulin Content • Daorongs data suggests that - 1. Insulin content in delta C treated cells is lower 2. Delta C treated cells secrete lower amounts of insulin when stimulated with secretagogues • My data suggests that 1. Global protein synthesis is reduced 2. Insulin synthesis is increased

  15. Next Step • More replicates • Check insulin content, too for untreated and vector treated controls for insulin content under similar conditions • IP another abundant protein

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