Skip to main content
. 2016 Feb 4;4:e1663. doi: 10.7717/peerj.1663

Figure 4. Receptor association of TLR4 and CD14 of PUFA-enriched and stimulated RAW264.7 macrophages.

Figure 4

Formation of TLR4-CD14 complexes was determined by co-immunoprecipitation (IP) and visualized by Western blot (WB). Cells were cultured in basic medium (RPMI 1640 containing 4.5 g/L glucose, 5% v/v FCS, 0.2% v/v ethanol) supplemented with alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) in a concentration of 15 µM for 72 h. Cell stimulation was performed by addition of LPS (1 µg/mL) or viable P. aeruginosa (MOI 1) to the culture medium in the last 24 h of incubation. TLR4-CD14 complexes from cell lysates were co-immunoprecipitated using an appropriate anti-mouse CD14 antibody and immunoblotted using an appropriate anti-mouse TLR4 antibody. Band size was checked using a protein ladder, and band intensities were analyzed densiometrically. The IP:CD14/WB:CD14 blot represents the lower part of the IP membrane and serves as loading control used for data normalization. To account for inter-assay variability, the stimulation/supplementation conditions compared were run on the same gel. Data from unstimulated (A) or unsupplemented (C+E+G) cells were set 100%, and values from stimulated (A) or supplemented (C+E+G) cells were expressed relative to this control. Four independent experiments were performed in duplicate for each combination of PUFA supplementation and stimulation of cells (N = 4, n = 2). (A) Graph showing the stimulus effects on TLR4-CD14 association of unsupplemented RAW264.7. (B) Representative Western blots showing the stimulus effects on TLR4-CD14 association of unsupplemented RAW264.7. (C) Graph showing the PUFA effects on TLR4-CD14 association of unstimulated RAW264.7. (D) Representative Western blots showing the PUFA effects on TLR4-CD14 association of unstimulated RAW264.7. (E) Graph showing the PUFA effects on TLR4-CD14 association of LPS-stimulated RAW264.7. (F) Representative Western blots showing the PUFA effects on TLR4-CD14 association of LPS-stimulated RAW264.7. (G) Graph showing the PUFA effects on TLR4-CD14 association of P. aeruginosa-stimulated RAW264.7. (H) Representative Western blots showing the PUFA effects on TLR4-CD14 association of P. aeruginosa-stimulated RAW264.7.