MALP-2 up-regulates TLR2/6 expression in endothelial cells. (A) TLR2 and TLR6 mRNA expression was determined by PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression is shown as loading control. Representative pictures of 3 independent experiments are shown. (B) TLR2 and TLR6 protein expression was demonstrated by Western blot. GAPDH expression is shown as loading control. Representative pictures of 3 independent experiments are shown. Human monocytic THP-1 cells were used as positive control. (C) Immunofluorescent staining of HUVECs with antibodies against TLR2 and TLR6. Representative pictures of 3 independent experiments are shown (Leica DM 4000B microscope, 40×/0.75 NA dry objective, Leica DFC 320 camera, Leica QWin Version 3 software). (D) Products of real-time PCR were run on agarose gels showing TLR2 and TLR6 mRNA levels in HUVECs after MALP-2 (1 μg/mL) stimulation for the indicated time points. β-Actin mRNA expression is shown as loading control. Representative pictures are shown. (E) Quantification of TLR2 and TLR6 mRNA levels relative to β-actin mRNA levels by real-time PCR (n = 10). *P < .05, **P < .01 vs 0 hours. (F) Western blot showing TLR2 and TLR6 protein levels in HUVECs after MALP-2 (1 μg/mL) stimulation for the indicated time points. GAPDH expression is shown as loading control. Representative pictures are shown. (G) Quantification of TLR2 and TLR6 protein levels relative to GAPDH protein levels (n = 4-6). *P < .05, **P < .01 vs 0 hours. (E,G) Error bars represent mean ± SEM.