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. 2011 Nov;31(21):4319–4334. doi: 10.1128/MCB.05033-11

Fig. 4.

Fig. 4.

Perinuclear location of LTβR is a prerequisite for the recruitment of endogenous TRAF proteins and induction of p100 processing. (A) Flow cytometry analysis of HEK 293 cells mock transfected or transfected with expression vector for LTβR ΔS wt, LTβR ΔS/ΔI 345–358, or LTβR ΔS/ΔI 359–368 and stained for cell surface LTβR (nonpermeabilized) or cell surface and intracellular LTβR (permeabilized). MFI (mean fluorescence intensity) represents the value of one measurement out of three independent experiments. (B) HeLa cells transiently transfected with the indicated HA-tagged construct were stained for LTβR (in red) and nuclei (DAPI). Arrows indicate the punctate perinuclear staining of LTβR. (C) HEK 293 cells were mock transfected or transfected with LTβR expression vectors encoding either wt, ΔI 345–358, ΔI 359–368, or their signal sequence (ΔS)-defective counterpart. Immunoprecipitated LTβR was analyzed by Western blotting for the recruitment of endogenous TRAF2 and TRAF3. The asterisks represent the cross-reactivity with Ig heavy chains. (D) Extracts from cells transfected with signal sequence (ΔS)-defective mutants were used to analyze the processing of p100 by Western blotting.