American Society of Hematology

Figure 7

$Figure 7. TRAF binding and degradation upon signaling via hCD40-P227A and WT hCD40. (A) M12.hCD40WT or M12.hCD40-P227A (P227A.1, P227A.2) cells were stimulated for 30 minutes with 1 Hi5: 5 B cells and cell lysates prepared. The Brij insoluble fraction (I) represents the lipid raft fraction, whereas the Brij soluble fraction (S) represents the non-raft portion of the cell lysates. hCD40 was immunoprecipitated as described27 and subjected to SDS-PAGE and Western blot analysis for TRAF6, followed by TRAF1 (not shown), TRAF2, TRAF3, and hCD40. Data are representative of 3 independent experiments. A second hCD40WT clone was tested with similar results (not shown). (B) CH12.hCD40WT (hCD40WT.1, hCD40WT.2) and CH12.P227A (P227A.1, P227A.2) were stimulated for the indicated times and cell lysates prepared. Membranes were immunoblotted for TRAF2, then stripped and reprobed for TRAF3 followed by GAPDH as a loading control. Data are representative of at least 3 experiments.$

TRAF binding and degradation upon signaling via hCD40-P227A and WT hCD40. (A) M12.hCD40WT or M12.hCD40-P227A (P227A.1, P227A.2) cells were stimulated for 30 minutes with 1 Hi5: 5 B cells and cell lysates prepared. The Brij insoluble fraction (I) represents the lipid raft fraction, whereas the Brij soluble fraction (S) represents the non-raft portion of the cell lysates. hCD40 was immunoprecipitated as described²⁷ and subjected to SDS-PAGE and Western blot analysis for TRAF6, followed by TRAF1 (not shown), TRAF2, TRAF3, and hCD40. Data are representative of 3 independent experiments. A second hCD40WT clone was tested with similar results (not shown). (B) CH12.hCD40WT (hCD40WT.1, hCD40WT.2) and CH12.P227A (P227A.1, P227A.2) were stimulated for the indicated times and cell lysates prepared. Membranes were immunoblotted for TRAF2, then stripped and reprobed for TRAF3 followed by GAPDH as a loading control. Data are representative of at least 3 experiments.

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