Figure 6.

Schematic representation of Nished and its C‐and N‐terminal deletion mutants. (A) The deletions were made and cloned in the mammalian expression vector, pcDNA6V5/HisB, as described in ‘Material and Methods’. Numbers denote the amino acids in the constructs. (B) microaffinity isolation of S³⁵ methionine labelled Nished and its N and C‐terminal deletion proteins generated by coupled transcription‐translation reaction with biotinylated CSS oligonucleotide as described in ‘Material and Methods’. (C) One tenth of reaction mixture volume of S³⁵ methionine labelled Nished and its N‐and C‐terminal deletions proteins were electrophoresed on an 18% SDS gel. (D) Functional analysis of Nished deletion mutants. Primary skeletal muscle cells were co‐transfected with the IRE mutant (pMutIRELuc and Nished or the N and C terminal deletions mutants). The luciferase activity was normalized against the internal control, Renilla luciferase activity. (n= 7 in triplicate, □ one sample t‐test with Bonferroni correction, P < 0.05). Nished full length protein; NΔ1 (34–137 a.a); NΔ2 (90137 a.a.); CΔ1 (1–64 a.a); CΔ2 (1–115 a.a); V, Vector alone (negative control); Nished 10X, competition with lysate contouring vector alone.