1 / 15

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
Download Presentation

Lab. Meeting (1 st August)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related