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. 2016 Jan 28;25(7):1392–1405. doi: 10.1093/hmg/ddw021

Figure 2.

Figure 2.

The C-terminal region of SMN binds both the N-terminal PRR/WW and the C-terminal catalytic domains of Itch. (A) Schematics of full-length SMN and SMN-deletion mutants (upper part). Empty pRK7 vector (mock) or FLAG-tagged SMN-deletion mutants were co-expressed with HA-Itch in 293T cells. SMN proteins were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting of FLAG and HA (lower gels). (B) Schematics of full-length Itch and Itch-deletion mutants (upper part). Empty pRK7 vector (mock) or HA-tagged Itch-deletion mutants were co-expressed with FLAG-SMN in 293T cells. Itch proteins were immunoprecipitated with an anti-HA antibody, followed by immunoblotting of FLAG and HA (lower gels). (C) HA-Itch (501–862) alone or with FLAG-SMN was expressed in 293T cells. HA-Itch (501–862) was immunoprecipitated with an anti-HA antibody, washed with buffers containing different concentration of NaCl, followed by immunoblotting of FLAG and HA. (D) Recombinant GST, GST-Itch or GST-Itch (501–862) was incubated with recombinant SMN for in vitro GST pull-down assays, similar to that shown in Figure 1D. Asterisk denotes residual BSA applied to reduce non-specific binding.