Fig 1.

Regulation of the NF-κB pathway by IκB proteins in U1 HIV latently infected cells. (A) IκB mRNA quantification by RT-PCR (left panel) and IκB protein quantitation by immunoblotting (right panel). In this figure, the RNA and protein values were normalized to the levels of IκBε. The dots indicate the different replicates, and the lines represent the mean values. (B) NF-κB mRNA quantitation by RT-PCR (left panel) and NF-κB protein monomer quantitation by immunoblotting (right panel). In this figure, the RNA and protein values were normalized to the levels of RelB. (C) Coimmunoprecipitation of the three canonical IκB proteins with p65, p52, and p50 NF-κB monomers. IκBα, IκBβ, and IκBε were immunoprecipitated with specific monoclonal antibodies, and the NF-κB proteins that coprecipitated with the IκB proteins were detected by immunoblotting using monoclonal antibodies against p65, p52, and p50, as listed in the figure. The positive-input control extract was electrophoresed using 10% of the extract subject to immunoprecipitation. The negative IgG control was rabbit IgG. IκBα interacted strongly with all NF-κB subunits and IκB proteins. IκBβ and IκBε interacted strongly with p65, but interacted less strongly with p52 and p50 compared to IκBα. (D) Degradation kinetics of the different canonical IκBs. Cells were treated with TNF-α (10 ng/ml), and protein was isolated after 0, 5, 15, 30, 60, 90, and 120 min. IκB proteins and an endogenous β-actin control protein were detected by SDS-PAGE and immunoblotting (left panel). The immunoblots were scanned and quantitated densitometrically, normalized to the endogenous β-actin control, and plotted (right panel). IκBα protein decreased rapidly to undetectable levels and stayed low for approximately 30 min, after which it increased until peaking at 90 min, at about twice the baseline level. IκBβ and IκBε had slower decay kinetics, reaching nadirs at about 15 min, but never becoming undetectable. After 60 min, both IκBβ and IκBε proteins exhibited a sustained increase, although none reached baseline levels by 120 min.