Question

Experiment

• The role of Apaf-1 protein in response to cellular stress induced by increased osmotic pressure outside the cells (hyperosmotic stress) was studied.
• Wild-type mouse embryonic fibroblasts (Apaf-1+/+cells) and homozygous Apaf-1 knock-out fibroblasts (Apaf-1–/–cells) were cultured in hyperosmotic medium for the time periods indicated in the figure.
• Protein extracts were prepared and subjected to Western blot analysis using anti-Apaf-1, anti-caspase-3, anti-eIF2α, and anti-tubulin antibodies, and an antibody specific for the phosphorylated form of eIF2a [labeled eIF2a-P(S51) in the figure].

Figure

The figure won't show up, but here is the link to the figure: https://oup-arc.com/access/content/cooper8e-student-resources/cooper8e-data-analysis-problem-19-4-the-role-of-apaf-1-protein-in-hyperosmotic-stress?previousFilter=all_resources

Questions

1. What was the goal of using anti-tubulin antibody?
2. How successful was the Apaf-1 knockout procedure?
3. According to the figure, describe the pathway by which caspase-3 is regulated during hyperosmotic stress.
4. What is the biological response of Apaf-1+/+ and Apaf-1–/– fibroblasts to hyperosmotic stress?

Answer 1

ans1. the anti tubulin antibody was used as control to show that experiment was proper and expression was not effected by the experimental error

2.Apaf knockout was successful because no Apaf expression was there in the Apaf-/- wells

3.caspase 3 is regualted by Apaf expression

Apaf-/-= doesn't show any caspase 3 expression

in Apaf+/+ caspase 3 is activated when hyperosmotic stress increases to 120 min or more then that

Apaf is essential for conversion of procaspase to caspase3

4.Apaf is influencing the expression of caspase3 which is involved in apoptosis

in Apaf+/+ the increased duration of hyperosmotic stress will lead to apoptosis which will be absent in Apaf-/-