Upregulation of TLR1, TLR2, TLR4, and IRAK-2 Expression During ML-1 Cell Differentiation to Macrophages: Role in the Potentiation of Cellular Responses to LPS and LTA
Figure 3
TPA induces upregulation of TLR1 and TLR4 proteins expression in human myeloid ML-1 cells. Three millions cells were incubated with 5 nM TPA for various times (24 hrs, 3 days, and 6 days). (a) Cells were then harvested, washed with PBS, fixed with formaldehyde (2%), permeabilized with ice methanol, and stained using (Alexa-Fluor-488-) labeled anti-human TLR1 or TLR4 antibodies. The levels of TLR1 or TLR4 protein expression in TPA-treated ML-1 cells were determined by FACS-Scan analysis. Negative controls were stained with isotype-matched- (Alexa-Fluor 488) conjugated IgG and compensation was adjusted using the single-stained cell samples. The fluorescence intensities were determined using Cellquest software (Becton Dickinson, Bedford, MA). Data were analyzed by ModFILT statistical software. The figure depicts representative results from one of three replicate experiments. (b) After transfer onto microscope slides, cells were washed with PBS, fixed with formaldehyde (2%), permeabilizes with PBS containing 0.25% Triton X-100, and stained using (Alexa-Fluor 488 labeled anti-human TLR1 or TLR4 antibodies). The levels of TLR1 or TLR4 protein expression were examined using fluorescent microscope.
