Journal: Infection and Immunity
Article Title: Involvement of endoplasmic reticulum and histone proteins in immunomodulation by TLR4-interacting SPA4 peptide against Escherichia coli
doi: 10.1128/iai.00311-23
Figure Lengend Snippet: Assessment of expression of calnexin, EMC1, LAMP1, and histone H2A in cells challenged with heat-killed E. coli 19138 and treated with SPA4 peptide. The timeline of infectious challenge and SPA4 peptide treatment is shown in (A). The whole-cell lysate protein (2–5 µg) was separated and immunoblotted with antibodies specific to calnexin, EMC1, LAMP1, and histone H2A proteins. The membrane then was stripped and re-probed for β actin. Lanes contained whole-cell lysate protein from unchallenged, vehicle-treated control (1), heat-killed E. coli-challenged, vehicle-treated (2), and heat-killed E. coli-challenged, SPA4 peptide-treated (3) JAWS II dendritic cells. Representative immunoblots for each protein are shown within the figure (B, i–iv). The densitometric units for immunoreactive bands were normalized with those of β actin. The ratios of arbitrary densitometric units are shown as bar (Mean ± SEM) and are derived from three experiments performed on separate occasions (C, i–iv). The fold changes (calculated as normalized value for respective protein in experimental group per unchallenged, vehicle control group) are shown as bar (Mean ± SEM) in (D, i–iv). The p values are shown within each figure (one-way ANOVA).
Article Snippet: The membrane with transferred proteins was immunoblotted with 1:500 or 1,000 diluted primary antibodies for calnexin (Abcam, Waltham, MA), EMC1 (Abclonal Technology, Woburn, MA), histone H2A (Cell Signaling Technology, Danvers, MA), and LAMP1 (Thermo Fisher Scientific, Waltham, MA) overnight and 1:500 or 1:1,000 diluted horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody.
Techniques: Expressing, Membrane, Western Blot, Derivative Assay
