Skip to main content
. 2018 Nov 9;7:e37545. doi: 10.7554/eLife.37545

Figure 3. SuperYan mutations increase SAMSAM affinity in vitro and full-length self-association in vivo.

(A) Representative sensogram of the wildtype Yan SAM interaction, via SPR. Concentrations of analyte are color-coded and listed to the right, and the kinetic model is represented by dashed lines. (B) Sensogram of the SY5 interaction, demonstrating stronger binding affinity. (C) Table of parameters obtained from kinetic fitting of sensograms for Yan and SY15 interactions. Two technical replicates were taken for each interaction to calculate parameters. (D) Co-immunoprecipitation of Yan and SY15 constructs. IPs are shown in the top panel, whereas lysate inputs are shown in the bottom panel. FLAG-tagged Yan constructs were immunoprecipitated and both HA-tagged and FLAG-tagged proteins were immunoblotted. Arrows mark the mobility of the Yan constructs, whereas asterisks mark unreduced IgG in the IP and a non-specific FLAG-reactive species in the lysate, respectively. Fold enrichment of IP to lysate relative to wildtype is noted.

Figure 3.

Figure 3—figure supplement 1. Yan A86D and V105R mutations can monomerize SuperYan, thereby allowing measurement of affinity.

Figure 3—figure supplement 1.

negGFP native gel assay for the proteins listed in the right-hand panel. All constructs run as monomers. The image that is shown is taken from three separate 4 – 15% gradient gels loaded and run in parallel.
Figure 3—figure supplement 2. SuperYan mutations increase SAMSAM affinity, as measured by SPR.

Figure 3—figure supplement 2.

(A–F) Two sensograms with kinetic model fits and residuals for the measured interactions. (A) Yan wild type. (B) SY1. (C) SY2. (D) SY3. (E) SY4. (F) SY5.
Figure 3—figure supplement 3. SuperYan mutations maintain interaction with wildtype Yan SAM interface.

Figure 3—figure supplement 3.

Pairwise co-immunoprecipitation of full length Yan and SY5, with the indicated constructs transfected in each lane. Heterotypic co-IP between WT and SY5 is observed in lanes 6 and 7. Arrows mark the mobility of Yan species, whereas asterisks mark unreduced IgG in the IP and a non-specific, FLAG-reactive species in the lysate. Note that the Yan signal is weak in lysates, but strong in the IP.