Fig 5. Characterisation of SP-1277 isoform.

(A) Schematic representation of the domain structure of SP-1277 isoform. Light blue bar shows the epitope for the N-terminal antibodies and the red bar shows epitope for the C-terminal antibodies. The nuclear localisation signal in the terminal region SP-1277N⁶²⁵is shown by a hashed blue bar. The boundary of SP-1277-N⁶²⁵ and SP-652 are shown two discontinuous lines. The SH3, DBL homology (DH) and PH domains are shown in SP-652. (B) Western blot analysis of the SPATA13 and its fragments analysed by N-terminus (ab122627) and anti-AcGFP antibodies. AcGFP fusion protein of SP-1277, SP-1277-N⁶²⁵ and the SP-652 were expressed in HT1080 cells, total cell lysate was separated on 4–15% SDS polyacrylamide gel, transferred on nitrocellulose membrane and probed with different antibodies. Specific reactivity with protein bands is shown by asterisks. (C) Western blot of total RPE-1 lysate probed with the C-terminus (lane 1) and N-terminal (lane 2, ThermoFisher; lane 3, Abcam) antibodies. (D) Reactivity of N- and C-terminal antibodies with RPE-1 cells overexpressing SP-1277 compared with AcGFP control. Immuohistochemical reactivity of murine eye tissues (E, F) and human eye tissues (G) with the N-terminus antibody from Abcam (ab122627). CE = ciliary epithelium, CP = ciliary process, PP = pars plana, GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer, SM = sphincter muscle, DM = dilator muscle, PE = posterior muscle. Re = retina, Co = cornea, Le = lens.