Activation of RAGE/TLR9 in the CLL microenvironment in vivo and in vitro. (A) Detection of RAGE and TLR9 expression in CLL cells by western blotting. Ten untreated and 6 relapsed CLL peripheral mononuclear cells were stored at −80°C and lysed directly from the pellets. Fifty micrograms of proteins was loaded onto each lane. Rabbit anti-RAGE antibody and mouse anti-TLR9 antibody were used at 1:3000 dilution. Hsp70 was used as a loading control. (B) Detection of RAGE and TLR9 expression in CLL-LN and RA-LN by immunohistochemical staining as described in the Materials and methods. Images were taken with a Leixa DM2500 microscope (original magnification, ×200). Immunofluorescent costaining of HMGB1/TLR9 and RAGE/TLR9 in (C) fresh CLL cells and differentiated NLCs after (D) 1 and (E) 2 weeks of in vitro culture. Costained primary antibodies used were mouse anti-TLR9 (red)/rabbit anti-HMGB1 (green) and mouse anti-TLR9 (red)/rabbit anti-RAGE (green) antibodies. Secondary antibodies for costaining were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 546 donkey anti-mouse IgG. Arrows in D show TLR9 and RAGE aggregation at the contacting sites with CLL cells. Empty and solid triangles in E indicate specified vesicles containing HMGB1/TLR9 and RAGE/TLR9. (F) Expression of HMGB1, RAGE, and TLR9. Proteins were extracted from fresh and 2-week cultured CLL cells and NLCs. Protein expression was detected by western blotting using mouse anti-HMGB1, anti-TLR9, or a rabbit anti-RAGE antibody. CD68 and vimentin were used as markers for NLCs, and β-actin was used for a loading control. (G) Co-IP was performed using mouse anti-TLR9 antibody, and the blots were probed with a rabbit anti-RAGE, rabbit anti-HMGB1, and mouse anti-TLR9 antibodies, respectively. This is a typical result from 3 individual experiments performed.