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 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  Comparison of beta emission generated signal from -Total cell lysates -(TCA precipitated) Total protein (from the cell lysates) : Gives data regarding percentage incorporation

Determination of time point of maximum S35 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)

S35 incorporation following 30 mins of pulsing Untreated lacZ ▲C

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

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

Immunoprecipitation for insulin Untreated LacZ ▲C Scintillation counter readings

Immunoprecipitation for insulin Untreated LacZ ▲C

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

Immunoprecipitation for insulin Reducing gel (with Urea)

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

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  More replicates  Check insulin content, too for untreated and vector treated controls for insulin content under similar conditions  IP another abundant protein