1. Identification of an unusual translation start in the published human MKL1.
Identification of an unusual translation start in the published human MKL1. (A) ATG-ΔC and 5′UTR-ΔC constructs were overexpressed in HEK293 cells, and MKL1 was detected by immunoblotting with anti-MKL1 total mAb. (B) Human 5′UTR-full-length MKL1 was overexpressed in HEK293 cells, the translated MKL1 was affinity purified, and the major product digested with AspN. Peptides were identified by LC-MS. Highlighted in gray, peptide coverage; black star, in-frame stop codon; green letters, suggested ATG, CTG or GTG translation initiation codons (coding for M, L and V, respectively); vertical arrow, putative GTG/Val-100 start. (C) Alignment of the human MKL1 5′UTR region with those of mouse (variant 2) and Xenopus. Horizontal arrows: 5′ to 3′ direction; black star, in-frame stop codon; vertical arrow, suggested GTG/Val-100 start; blue 4-point star, nucleotides complying with the Kozak consensus sequence. (D) Point mutations targeting the suggested GTG/Val-100 start codon were introduced into the construct shown in A, the resulting constructs were transiently overexpressed in HEK293 cells and MKL1 was detected by immunoblotting with anti-MKL1 total mAb. Endogenous human MKL1 was purified from untransfected (Untransf.) HEK293 cells as described in B.
Matthias A. Scharenberg et al. J Cell Sci 2014;127:
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