Quantifying RIP2 Protein Expression in ETEC-Treated HT29 Cells

Cell lysates were harvested from siRNA-treated HT29 cells after treatment with or without ETEC WT OMVs (final concentration of 100 ng/mL protein equivalent). Cells were briefly rinsed with PBS and lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl, 150 mM NaCl, 1.0% [vol/vol] NP-40, 0.5% [wt/vol], sodium deoxycholate, 1.0 mM EDTA, 0.1% [wt/vol] SDS, 0.01% [wt/vol] sodium azide [pH 7.4]). The protein concentration in the lysate was determined using the Bradford assay (with Bio-Rad Laboratories protein assay dye reagent) according to the manufacturer’s manual. Lysates were separated by SDS-PAGE using 12% gels and the PageRuler prestained protein ladder (Thermo Fisher Scientific) as a molecular mass standard. Ten micrograms of each sample was loaded and transferred to a nitrocellulose membrane (Amersham) for immunoblot analyses, which were essentially performed as described previously (97 (link)). Anti-RIP2 antibody (abcam, ab8428) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14C10) antibody (Cell Signaling; 2118S) were used in combination with the HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch; 111-035-003) as the primary and secondary antibodies, respectively.