Journal: Biomedicines
Article Title: TIRAP/Mal Positively Regulates TLR8-Mediated Signaling via IRF5 in Human Cells
doi: 10.3390/biomedicines10071476
Figure Lengend Snippet: Silencing of TIRAP gene inhibits nuclear translocation of IRF5 in 60 min after CL075 stimulation. Experiments were performed on human MDMs ( n = 6 donors). ( a ) TIRAP -silencing efficacy was quantified with RT-qPCR from parallel wells of CL075 stimulated cells using non-stimulated cells for normalization (fold = 1.0). Control or TIRAP -silenced cells were stimulated with CL075 (2 μg/mL) for one hour, followed by fixation of cells, double staining of IRF5 ( b ) and NF-kB (p65/RelA) ( c ), DNA staining by Hoechst 3342 for nuclei visualization, and quantitative imaging by high-content screening (Olympus Scan^R system). The level of nuclear IRF5 ( b ) and p65 ( c ) was calculated as the percentage of positively stained nuclei multiplied by the mean fluorescence-intensity value (MFI) of the positively stained nuclei. In non-stimulated cells, the background-staining levels (%pos × MFI) for nuclear IRF5 and p65 were <15 and <73, respectively. ( d , e ) Representative immunofluorescent images of non-stimulated (NS) and CL075 stimulated cells used for quantification of IRF5 (red channel) and p65 (green channel) in nuclei (blue channel). Scale bar shown in overlay represents 50 µm. Statistical significance was examined with paired t -test (** p < 0.01, **** p < 0.001).
Article Snippet: The following primary antibodies were used: mouse GAPDH (ab9484), rabbit β-tubulin (ab6046) from Abcam (Cambridge, UK); rabbit phospho-Akt Ser473 (D9E XP), phospho-p38 MAPK (T180/Y182), phospho-STAT1 (Tyr701) (58D6), phospho-TAK1 (T184/187) (90C7), phospho-JNK (81E11) (T183/Y185), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), IκBα (44D4), IRAK1 (D51G7), MyD88 (D80F5), Histone H3 (3H1), and phospho-NF-κB p65 (Ser536) (93H1) from Cell Signaling Technology (Danvers, MA, USA); rabbit PCNA Abs were from Santa Cruz Biotech (Santa Cruz, CA, USA); sheep IRF5 and IRAK4 were from MRC-PPU Reagents (University of Dundee, Dundee, UK); goat TIRAP polyclonal Abs were from Invitrogen (#PA5-18439, Waltham, MA, USA), and mouse STAT1 antibodies were from BD Biosciences (#610185, Wokingham, UK).
Techniques: Translocation Assay, Quantitative RT-PCR, Control, Double Staining, Staining, Imaging, High Content Screening, Fluorescence
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