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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 - PowerPoint PPT Presentation
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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
0
5
10
15
20
25
30
35
Time
Perc
enat
ge
Inco
rpor
atio
n
Series1
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
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
Untreated LacZ ▲C
total protein
labeled fraction
total protein
labeled fraction
total protein
labeled fraction
0
500
1000
1500
2000
2500
comparison total protein vs labeled protein
total protein
labeled fraction
Autoradiograph for a western of total protein (TCA precipitated samples) to check for signal
Immunoprecipitation for insulin
IP
Total protein
IP
Total protein
IP
Total protein
02000000400000060000008000000
10000000120000001400000016000000
CPM
IPed protein as a fraction of total labeled protein
IP
Total protein
Untreated LacZ ▲C
Scintillation counter readings
Immunoprecipitation for insulin
Iped protein(Ins)
0.62
0.63
0.64
0.65
0.66
0.67
0.68
Percent
Iped Insulin as a percentage
Iped protein(Ins)
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
ImageQuant
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Avera
ge s
ign
al/
are
a
ImageQuant
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
