A 35-aa region within the ζ-chain is responsible for its degradation after repeated LPS treatment. (A) RNA was extracted from spleens of control and LPS-treated mice and was analyzed by Northern blot. ζ-mRNA was detected after hybridization with a ³²P-labeled ζ-specific cDNA probe (top panel). Hybridization with a ³²P-labeled CD3ε-specific cDNA probe was used as a loading control (top panel). Densitometric analysis is shown as the ratio of ζ-chain to CD3ε mRNA (bottom panel). (B) Freshly isolated control and LPS-treated splenocytes were treated with or without bafilomycin A1 (1 μM or 0.5 μM) and then analyzed for total ζ-chain expression by FACS. The results are presented as the mean fluorescence intensity (MFI) of 2 independent experiments, and standard deviations are shown. (C) Representative density plots (left panel) of Gr-1⁺Mac-1⁺ cell distribution within the spleens of control and LPS-treated ζ-transgenic mice (described in panel D). Splenic T cells (Thy1.2⁺ cells) from LPS-treated ζ-transgenic mice were analyzed for ζ-chain expression levels by FACS (right panel). (D) Schematic representation of ζ-chain sequences in transgenic mice expressing FL and truncated ζ-chain (distal and TL). The extracellular (EC), transmembrane (TM), and cytoplasmic domains are depicted, and the location of ITAMs (I-III) in the cytoplasmatic domains of the ζ proteins and their tyrosine residues (Y) are indicated (D indicates deletion). A double-headed arrow represents the 35-aa ζ-chain region responsible for its targeting to lysosomal degradation. The aa sequence of this region is shown. A schematic representation of truncated ζ-chain (proximal) sequence, which lacks the above-mentioned 35-aa area. (E) ζ-deficient hybridoma T cells (MA5.8) were stably transfected with FL or truncated ζ-chain (proximal) as described in Materials and methods. T cell–transfected hybridoma T cells (2 × 10⁵) were coincubated for 16 hours with 4 × 10⁶ splenocytes derived from control (gray area) or LPS-treated (black line) B10.A mice. The hybridoma T cells were then analyzed for total CD3ε and ζ-chain expression levels by FACS.