Lab. Meeting (18th 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

S35 Incorporation: Total Protein

0

5

10

15

20

25

30

35

Time

Perc

enat

ge

Inco

rpor

atio

n

Series1

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

0

5

1015

20

25

3035

40

45

% incrprn.

s35 incrprn final & revised

Untreated lacZ ▲C

Labeling as a fraction of total Protein content in samples

0200400600800

10001200140016001800

ug/800ul cell lysate

labelled fraction totalprtn.n

total protein

Untreated LacZ ▲C

Autoradiograph for a western of the same

Next step

Immunoprecipitation for insulin Comparing Insulin levels from different cell

lysate samples