Download Shounak_080105

Lab. Meeting
(1st 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
S35 Incorporation: Total Protein
35
Series1
Percenatge
Incorporation
30
25
20
15
10
5
0
Time
Studying protein synthesis (intracellular
compartment) using S35 labeling
 832/13 cells
= Untreated controls
 832/13 cells with lacZ
= (adenovirus vector without
the dominant negative
construct)
 832/13 cells with ▲C
=(adenovirus vector with
the dominant negative
construct)
INCUBATED for 36 hours, no significant cell death
at the time of harvesting
S35 incorporation following 30 mins of pulsing
s35 incrprn final & revised
45
40
35
30
25
% incrprn.
20
15
10
5
0
Untreated
lacZ
▲C
Labeling as a fraction of total Protein content in
samples
comparison total protein vs labeled protein
2500
total protein
2000
total protein
1500
total protein
total protein
labeled fraction
1000
labeled fraction
500
labeled fraction
labeled fraction
0
Untreated
LacZ
▲C
Autoradiograph for a western of total protein
(TCA precipitated samples) to check for signal
Immunoprecipitation for insulin
IPed protein as a fraction of total labeled protein
16000000
Total protein
14000000
12000000
10000000
CPM 8000000
6000000
4000000
2000000
0 IP
Untreated
Total protein
Total protein
IP
Total protein
IP
LacZ
IP
▲C
Scintillation counter readings
Immunoprecipitation for insulin
Iped Insulin as a percentage
0.68
0.67
0.66
Percent 0.65
Iped protein(Ins)
0.64
0.63
0.62
Iped protein(Ins)
Untreated
LacZ
▲C
Immunoprecipitation for insulin
Non Reducing gel (without Urea)
Untreated
LacZ
▲C
Non Specific Aggregates?
Immunoprecipitation for insulin
Reducing gel (with Urea)
Immunoprecipitation for insulin
ImageQuant
0.8
0.7
Average signal/area
0.6
0.5
0.4
ImageQuant
0.3
0.2
0.1
0
Untreated
LacZ
▲C
Suggests higher insulin synthesis in delta C treated cells
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