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. Author manuscript; available in PMC: 2013 Nov 15.
Published in final edited form as: J Immunol. 2010 Sep 24;185(9):10.4049/jimmunol.1001653. doi: 10.4049/jimmunol.1001653

Figure 1. Ubiquitinated Ag-BCR Complexes are Restricted to Lipid Raft Membrane Domains.

Figure 1

A20μWT B cells were surface “tagged” with either CTB-HRP (to label GM1+ lipid rafts) or anti-huIgM-btn (to label surface human IgM BCR molecules). The cells were then lysed in cold 0.1% TX-100 and lipid rafts separated from non-rafts by sucrose density gradient centrifugation (5, 6). Panel A – The refractive index, total protein and CTB-HRP level in each gradient fraction was determined. The raft region of the gradient is at the first inflection point in the refractive index curve, where CTB-HRP is exclusively found. The non-raft region of the gradient is the bottom 1/3 of the gradient (fractions 16 −24), which has a uniformly high refractive index and protein content. Panel B – Samples from pools of 100% of raft fractions and ~30% of non-raft fractions from the anti-huIgM-btn tagged cells (incubated for either 5 or 20 minutes at 37°C after tagging) were directly probed by western blot with SA-HRP to detect “Total” ligand-BCR complexes. Ubiquitinated ligand-BCR complexes were isolated from the remainder of the pools by UQ1 pull-down and detected by blotting with SA-HRP (14). Shown is a representative result from 1 of 5 independent experiments.