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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

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lab meeting 1 st august
Lab. Meeting(1st August)
  • In Vitro studies to define the role of PERK in insulin synthesis

* Using the 832/13 cell line

experimental design
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)

determination of time point of maximum s35 incorporation
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

studying protein synthesis intracellular compartment using s35 labeling
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
autoradiograph for a western of total protein tca precipitated samples to check for signal
Autoradiograph for a western of total protein (TCA precipitated samples) to check for signal
immunoprecipitation for insulin
Immunoprecipitation for insulin

Untreated LacZ ▲C

Scintillation counter readings

immunoprecipitation for insulin non reducing gel without urea
Immunoprecipitation for insulinNon Reducing gel (without Urea)

Untreated LacZ ▲C

Non Specific Aggregates?

immunoprecipitation for insulin2
Immunoprecipitation for insulin

Untreated LacZ ▲C

Suggests higher insulin synthesis in delta C treated cells

insulin content
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

next step
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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