HSP110 intervenes upstream of the IRAK1-TRAF6 complex formation. (A) In cellulo interaction of IgM with p-IκBα was determined in HBL1, OCI-Ly3, and U2932 by Duolink technology after transfection with an siRNA targeting HSP110 or a control siRNA (n = 3; 1 representative image is shown). Scale bar represents 20 μm. (B) Quantitation of IgM–p-IκBα spots per cell determined by Duolink technology as in (A). (C) Immunoblot analysis of HSP110, p-IRAK1, and total IRAK1 in HBL1, OCI-Ly3, and U2932 cells at 48 hours after transfection with an siRNA targeting HSP110 or a control siRNA. β-actin served as a loading control (n = 3). (D) Immunoprecipitation of Lysine 63–linked polyubiquitin chain (K63-ub) in SU-DHL2 and OCI-Ly10 cells 48 hours after transfection with an siRNA targeting HSP110 or a control siRNA, followed by immunoblot using a TRAF6 antibody. Immunoblot of total cell extract (input) shows HSP110 and TRAF6 analysis (n = 3). ***P < .001.