HSP110 interacts with MyD88 to enhance NF-κB signaling. (A) Immunoblot analysis of p-IκB, IκB, HSP110, and MyD88 in Burkitt lymphoma (BJAB) cells 48 hours after transfection with plasmids coding for HSP110-GFP, and/or MyD88 WT, or MyD88 L265P. HSC70 served as a loading control (n = 3). (B) Luciferase reporter experiment in HEK293T cells. Cells co-transfected with GFP reporter (internal control) and NF-κB luciferase reporter plasmid with or without increasing concentrations of untagged HSP110 and with or without 0.4 μg of Myd88 L265P or Myd88 WT plasmids (n = 3). Shown are relative luciferase values (luciferase:GFP ratio). Data are presented as the mean ± standard deviation. (C) In cellulo interaction of HSP110 with MyD88 was determined in HBL1, OCI-Ly3, and U2932 by Duolink technology in the presence or not of siRNA HSP110. HSP70 was used as a positive control for HSP110 interaction (1 representative image is shown for each condition). Scale bar represents 20 μm. (D) Quantitation of HSP110-HSP70 and HSP110-MyD88 spots per cell determined by Duolink technology as in (A). Boxplots depict the mean (line), the 25-75 percentiles (box) and the 10-90 percentiles (whiskers). (E) Immunoprecipitation (IP) of HSP110 and MyD88 in SU-DHL2 and OCI-Ly3 cells followed by immunoblot using an anti-MyD88 and anti-HSP110 antibody. A nonrelevant antibody was used as a control (IP control) (n = 3). (F) Immunoprecipitation of HSP110-GFP (IP-GFP) and MyD88-HA (IP-HA) in HEK293 cells 48 hours after transfection with plasmids coding for HSP110-GFP with or without MyD88-HA L265P (0.7 µg) or MyD88-HA WT (0.7 µg) (n = 3). Endo, endogenous. **P < .01; ***P < .001.