Fig. 4
From: Structural basis of SALM5-induced PTPδ dimerization for synaptic differentiation
Structure-guided biophysical and functional characterizations of the SALM5/PTPδ interaction. a Upper panel: SDS-PAGE analysis of SALM5 LRR-Ig (labeled as SALM5 for short) wild-type (WT) and its variants L327N, E365N, and K309N/R311T pulled down by Fc-tagged PTPδ Ig1–3 (labeled as PTPδ-Fc for short); lower panel: SDS-PAGE analysis of proteins used in the pull-down assay. M: molecular weight marker (kDa). b SPR sensorgrams of immobilized Fc-tagged PTPδ Ig1–3 (MeA−) binding to SALM5 LRR-Ig wild-type (WT) and its variants L327N, E365N, and K309N/R311T. K d values for WT and L327N are, respectively, 281.8 and 145.3 nM, whereas K d values for mutants of E365N and K309N/R311T were not determined due to impaired SALM5/PTPδ interactions. c Representative images of HEK293T cells displaying Myc-tagged PTPδ Ig1–3 (MeA−) incubated with vehicle (control) and recombinant His-tagged proteins of SALM5 LRR-Ig WT and its variants L327N, E365N, and K309N/R311T, and then immunostained with antibodies against Myc-tag (red) and His-tag (green). Scale bar, 20 μm. d Representative images of neurons co-cultured with HEK293T cells expressing the WT full-length SALM5 ectodomain (WT) and its mutant K309N/R311T. Scale bar, 20 μm. Immunostaining and synapsin-I intensity measurement were following the protocol used for Fig. 2e. e Quantification of the result in d. n = 14 fields of view, ***p < 0.005 for WT vs mutant, Student’s t test. f Representative images of synapsin-I (red) clustering in neuron co-cultured with HEK293T cells that were transfected with plasmids for empty pDisplay vector, L327N and E365N mutants of the full-length SALM5 ectodomain. Scale bar, 25 μm. Images for HEK293T cells were taken in bright field since mutants L327N and E365N were not expressed and no HA-tagged protein was displayed on the surface of HEK293T cells (Supplementary Fig. 7). In the bright field, the locations of primary neurons are indicated using yellow arrows. g Quantification of the result in d, n = 14 fields of view. Error bars in e and g present standard error of the mean (S.E.M.)
