Proteins uniquely associated with ARC-purified hmCD40 as determined by mass spectrometry analysis.

<p>*All protein assignments listed met or exceeded the Scaffold 95% confidence filter.</p><p>**TRAF1 was detected in gel slices containing material at or above the known molecular weight for TRAF1 in hmCD40 ARC samples, but was also detected in both hmCD40Δ67 and hmCD40 gel slices containing material of approximately 35 kD.</p

Western blot verification of mass spectrometry results.

<p>hmCD40 and hmCD40Δ67 ARC samples (3.3×10⁶ cell equivalents per lane) were fractionated by SDS-PAGE and transferred to PVDF membrane for Western blotting with the indicated antibodies. Lanes containing whole cell lysates (1.0×10⁵ cell equivalents per lane) were run in parallel. Each blot is representative of three or more experiments.</p

TRAF-dependent recruitment of proteins to CD40.

<p>Endogenous CD40 from CH12.LX cells (WT), TRAF2-deficient CH12.LX (T2-), or TRAF3 deficient CH12.LX cells (T3-) was isolated using ARC (lane loading as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011380#pone-0011380-g002" target="_blank">Fig. 2</a>). Western blotting was performed with the indicated antibodies. Each blot is representative of three or more experiments.</p

HOIPΔRBR inhibits IκBα phosphorylation and degradation.

<p>TRAF6-deficient A20.2J cells were stably transfected with FLAG2X-tagged HOIP or HOIPΔRBR in an IPTG-inducible expression vector. A, Expression of FLAG2X-tagged HOIP or HOIPΔRBR in stably transfected cell lines. Western blots of cell lysates from uninduced and IPTG-induced cell lines were probed with anti-FLAG (left) and anti-HOIP (right). Blots were reprobed for actin to verify equivalent lane loading. B, The two cell lines were stimulated with CD154 (CD40 ligand) for the times indicated and then processed for Western blotting. Unstimulated cells (first lane) and cells incubated for 5 minutes with insect cells lacking CD154 served as negative controls. Western blotting for phosphorylated IκBα was performed; blots were then stripped and reprobed for total IκBα and actin. C, Quantification of IκBα degradation (panel B) in TRAF6-deficient A20.2J cells stably transfected with Lac repressor only (◊;♦), Lac repressor plus full-length HOIP (□;▪), or Lac repressor plus HOIPΔRBR (Δ; ▴). Filled symbols indicate cultures pre-treated with IPTG. IκBα bands in each lane were normalized to the actin signal. The degradation index is the fraction of IκBα remaining at each time point relative to the amount of IκBα present in cells incubated for 5 minutes with insect cells lacking CD154. The results presented are the mean of four experiments. Error bars indicate standard error of the mean.*, p<0.05 (one-sided Student's t test).</p