Lab. Meeting (18 th July)  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  Equal amounts of S35 was added to each dish  The cell lysates were harvested at different time points (This would be indicative of radiolabelling in the intracellular compartment)

Determination of time point of maximum S35 incorporation

Studying protein synthesis (intracellular) 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 the same

Next step  Immunoprecipitation for insulin  Comparing Insulin levels from different cell lysate samples