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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 precipitated samples) to check for signal

Untreated LacZ ▲C

Scintillation counter readings


Immunoprecipitation for insulin1
Immunoprecipitation for insulin precipitated samples) to check for signal

Untreated LacZ ▲C


Immunoprecipitation for insulin non reducing gel without urea
Immunoprecipitation for insulin precipitated samples) to check for signal Non Reducing gel (without Urea)

Untreated LacZ ▲C

Non Specific Aggregates?


Immunoprecipitation for insulin reducing gel with urea
Immunoprecipitation for insulin precipitated samples) to check for signal Reducing gel (with Urea)


Immunoprecipitation for insulin2
Immunoprecipitation for insulin precipitated samples) to check for signal

Untreated LacZ ▲C

Suggests higher insulin synthesis in delta C treated cells


Insulin content
Insulin Content precipitated samples) to check for signal

  • 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 precipitated samples) to check for signal

  • 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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