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

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?

A) A noncovalent protein-protein interaction
B) A covalent protein-protein interaction
C) A noncovalent protein-carbohydrate interaction
D) A covalent protein-carbohydrate interaction

Correct Answer:

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