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. 2017 Jan 25;7(3):983–989. doi: 10.1534/g3.116.036939

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Copyright © 2017 Baek et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Verification of expression of selected misannotated and unannotated sORFs and small “y” genes by western blot. The sORFs and small genes examined for their expression are grouped into three categories: (A) Salmonella-specific, (B) conserved in Enterobacteriaceae, and (C) unassigned. “Unassigned” indicates sORFs whose conservation could not be determined by tBLASTn searches due to their short amino acids lengths. Mutant strains each carrying a chromosomal SPA tag fused to the C terminus of a target sORF/small gene were grown in respective medium used for ribosome profiling experiments (see Materials and Methods). Whole cell extracts (equivalent to the cell number at OD₆₀₀ 0.05) were run on a 16.5% SDS-PAGE gel, and the expression of SPA-tagged peptides/proteins was determined by western blot using an alkaline phosphatase-conjugated anti-FLAG antibody. A negative value on the y axis (ribo or mRNA density) indicates genes are located on reverse strand. The positions of the markers are shown for the approximate sizes of proteins (kDa). As a negative and a positive control for western blot, the whole cell extracts of the wild-type (no SPA tag) and wild-type cells expressing only SPA tag (tag only) were used (D).

Verification of expression of selected misannotated and unannotated sORFs and small “y” genes by western blot. The sORFs and small genes examined for their expression are grouped into three categories: (A) Salmonella-specific, (B) conserved in Enterobacteriaceae, and (C) unassigned. “Unassigned” indicates sORFs whose conservation could not be determined by tBLASTn searches due to their short amino acids lengths. Mutant strains each carrying a chromosomal SPA tag fused to the C terminus of a target sORF/small gene were grown in respective medium used for ribosome profiling experiments (see Materials and Methods). Whole cell extracts (equivalent to the cell number at OD₆₀₀ 0.05) were run on a 16.5% SDS-PAGE gel, and the expression of SPA-tagged peptides/proteins was determined by western blot using an alkaline phosphatase-conjugated anti-FLAG antibody. A negative value on the y axis (ribo or mRNA density) indicates genes are located on reverse strand. The positions of the markers are shown for the approximate sizes of proteins (kDa). As a negative and a positive control for western blot, the whole cell extracts of the wild-type (no SPA tag) and wild-type cells expressing only SPA tag (tag only) were used (D).