Passage
The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting. In addition, the protein insulin receptor substrate 4 (IRS4) , known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4. Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection. HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively) . Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away. The bound proteins were then visualized by Western blot using α-myc antibodies. The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb) . Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1.
Figure 1 Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub) . The results are shown in Figure 2.
Figure 2 Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control) , myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid
Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95.
-Which of the following most accurately describes the interaction between α-flag and flag-IRS4?

Question

Passage
The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting. In addition, the protein insulin receptor substrate 4 (IRS4) , known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4. Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection. HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively) . Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away. The bound proteins were then visualized by Western blot using α-myc antibodies. The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb) . Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1.

Figure 1 Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub) . The results are shown in Figure 2.

Figure 2 Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control) , myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid
Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95.
-Which of the following most accurately describes the interaction between α-flag and flag-IRS4?

Quizplus · Accepted Answer

11ecba2d_0efc_40ad_a3bf_81496b385e02_MD0008_00 Antibodies are proteins that function in the immune system to recognize foreign particles known as antigens. Specifically, antibodies mark foreign antigens for destruction (eg, phagocytosis) by other immune cells. Each antibody contains a variable region that binds to a specific chemical structure within an antigen, known as an epitope. Typically, the epitope is a sequence of amino acids within a protein or peptide. Antibodies bind their epitopes through noncovalent interactions such as hydrogen bonding and electrostatic attractions. The passage states that cell lysates were incubated with α-flag, an antibody that binds the flag peptide. Therefore, flag was the epitope, and it was this portion of flag-IRS4 to which the antibody noncovalently bound. Because the antibody and flag-IRS4 are both proteins, the binding interaction can be classified as a noncovalent protein-protein interaction. (Choice B) Covalent interactions occur when atoms share electrons. Antibodies generally bind their epitopes through noncovalent interactions such as hydrogen bonds and ionic interactions. (Choices C and D) Antibodies and IRS4 are both proteins, so they bind through protein-protein interactions rather than protein-carbohydrate interactions. Educational objective: Antibodies are proteins that noncovalently bind specific chemical groups on proteins known as epitopes. Epitopes are typically portions of proteins or peptides, so antibodies typically participate in noncovalent protein-protein interactions.

Passage The Ankyrin Repeat and SOCS Box Containing Protein 4 (Asb-4)