MCDB165a 2024 Midterm 2
1. You are a Caltech scientist studying a signaling pathway in mouse fibroblasts. When a G
protein coupled receptor is activated by a really big bang, you hypothesize that there will be
an increase in transcription of downstream genes that will result in higher protein levels of the
SHELDON protein. Assuming you have a method to generate a really big bang, and other
reagents that you require, explain how you would test your hypothesis that a big bang will
result in higher levels of the Sheldon protein. (No more than 6 sentences)(10 points)
I would have a tissue culture plate of mouse fibroblasts that I subject to a really big bang and a
control plate that I do not treat with a big bang. I would isolate protein lysates from the cells on
the 2 plates. I would use a western blot to monitor the levels of Sheldon in the big bang-treated
and control samples. I would determine whether Sheldon levels increase by western blot in the
big bang-treated cells compared with the untreated cells.
2. In the following figure from Park et al, Fig 2B
a. Why are there no bands in the first lane, the vector lane? (No more than 3 sentences.)
(5 points)
There are no bands because the sterile 5 with the HA tag was not introduced, so the anti-HA
antibody did not pull anything down.
b. Why is there no band in the Ste11-nNos blot in the second lane, the Ste5*-HA lane?(No
more than 3 sentences.)(5 points)
In the second lane, Ste5* is mutated so that Ste11 doesn’t bind to it. This Ste5*-HA doesn’t
have a syn, so Ste11-nNOS does not bind.
c. Why is there a band in the Ste11-nNOS blot in the fourth lane, the Ste5-HA lane?(No
more than 3 sentences)(5 points)
, There is a band in the Ste11-nNOS blot iin the fourth lane because Ste5 can bind to Ste11.
Immunoprecipitating with an HA antibody brings down Ste5-HA, and, because it binds to Ste11,
also Ste 11.
d. Why is there a band in the Ste11-nNOS blot in the third land, the Ste5*-syn-HA lane?
(No more than 3 sentences)(5 points)
There is a band in the sterile-11 nNOS blot in the third lane because the syn from Ste5*-syn-HA
binds the NOS fused to sterile 11. The antibody against HA pelleted Ste5*-syn-HA and the
pellet includes Ste11-nNOS. The two proteins interact because of the syn-notch interactions.
3. In the following figure, Fig 3c from Spira,
a. What was the experiment that the authors performed and what is plotted here?(No
more than 6 sentences)(8 points)
The authors generated reporters with GFP fused to a yeast protein that localizes to the plasma
membrane. The performed fluorescence recovery after photobleaching and monitored the rate
that the proteins moved into the bleached out area. They compared the half life for the
recovery to the number of transemembrane domains in the proteins that have transmembrane
domains and plotted that.
b. What did the authors conclude?(No more than 3 sentences)(6 points)
They concluded that there is no correlation so the number of times a protein crosses the
membrane is not a strong determinant of the rate that it moves.
1. You are a Caltech scientist studying a signaling pathway in mouse fibroblasts. When a G
protein coupled receptor is activated by a really big bang, you hypothesize that there will be
an increase in transcription of downstream genes that will result in higher protein levels of the
SHELDON protein. Assuming you have a method to generate a really big bang, and other
reagents that you require, explain how you would test your hypothesis that a big bang will
result in higher levels of the Sheldon protein. (No more than 6 sentences)(10 points)
I would have a tissue culture plate of mouse fibroblasts that I subject to a really big bang and a
control plate that I do not treat with a big bang. I would isolate protein lysates from the cells on
the 2 plates. I would use a western blot to monitor the levels of Sheldon in the big bang-treated
and control samples. I would determine whether Sheldon levels increase by western blot in the
big bang-treated cells compared with the untreated cells.
2. In the following figure from Park et al, Fig 2B
a. Why are there no bands in the first lane, the vector lane? (No more than 3 sentences.)
(5 points)
There are no bands because the sterile 5 with the HA tag was not introduced, so the anti-HA
antibody did not pull anything down.
b. Why is there no band in the Ste11-nNos blot in the second lane, the Ste5*-HA lane?(No
more than 3 sentences.)(5 points)
In the second lane, Ste5* is mutated so that Ste11 doesn’t bind to it. This Ste5*-HA doesn’t
have a syn, so Ste11-nNOS does not bind.
c. Why is there a band in the Ste11-nNOS blot in the fourth lane, the Ste5-HA lane?(No
more than 3 sentences)(5 points)
, There is a band in the Ste11-nNOS blot iin the fourth lane because Ste5 can bind to Ste11.
Immunoprecipitating with an HA antibody brings down Ste5-HA, and, because it binds to Ste11,
also Ste 11.
d. Why is there a band in the Ste11-nNOS blot in the third land, the Ste5*-syn-HA lane?
(No more than 3 sentences)(5 points)
There is a band in the sterile-11 nNOS blot in the third lane because the syn from Ste5*-syn-HA
binds the NOS fused to sterile 11. The antibody against HA pelleted Ste5*-syn-HA and the
pellet includes Ste11-nNOS. The two proteins interact because of the syn-notch interactions.
3. In the following figure, Fig 3c from Spira,
a. What was the experiment that the authors performed and what is plotted here?(No
more than 6 sentences)(8 points)
The authors generated reporters with GFP fused to a yeast protein that localizes to the plasma
membrane. The performed fluorescence recovery after photobleaching and monitored the rate
that the proteins moved into the bleached out area. They compared the half life for the
recovery to the number of transemembrane domains in the proteins that have transmembrane
domains and plotted that.
b. What did the authors conclude?(No more than 3 sentences)(6 points)
They concluded that there is no correlation so the number of times a protein crosses the
membrane is not a strong determinant of the rate that it moves.
