Journal: Journal of Inflammation (London, England)
Article Title: Lipopolysaccharide pretreatment increases the sensitivity of the TRPV1 channel and promotes an anti-inflammatory phenotype of capsaicin-activated macrophages
doi: 10.1186/s12950-024-00391-0
Figure Lengend Snippet: Capsaicin-induced calcium influx and modulation of LPS-induced signaling cascades. The kinetics of capsaicin-induced calcium mobilization in J774 cells were measured by the fluorescence of FLUO4-NW ( a ). Data represent mean values ± SEM. The level of statistical significance was determined using 2-way ANOVA followed by Tukey’s test for multiple comparisons (**** p < 0.0001). For detection of LPS-induced signaling cascades J774 were preincubated for 4 h and stimulated with capsaicin (C) or LPS (L) for 5–10 min. The ratio between phosphorylated and unphosphorylated p-38 ( b ) and ERK 1/2 ( c ) was obtained by Western blotting after normalizing the intensities of the p-p38 and p-ERK1/2 bands to those of total p38 and ERK1/2, respectively. Uncropped images of the western blot membranes are also shown in Supplementary Fig. and . Data are shown as boxes and whiskers. The lines within the boxes represent the median, and the whiskers represent the minimum and maximum values. n = 6 ( a-c ). Representative western blots are shown. The percentage of cells in which ERK1/2 translocated to the nucleus was determined by image flow cytometry. n = 3 ( d ). Data are shown as means ± SD. The level of statistical significance was determined using one-way ANOVA followed by Tukey’s test for multiple comparisons ( n p < 0.05, nn p < 0.01, nnn p < 0.001, nnnn p < 0.0001). # indicates the significance from (– –) group. + indicates significance from (C –) group. Other statistical significances between groups are indicated by *
Article Snippet: The nitrocellulose membrane was incubated with the following primary antibodies against extracellular signal-regulated kinase (ERK)1/2 (137F5; Cell Signaling, Danvers, MA, USA), p-ERK1/2 (197G2; Cell Signaling), p38 (sc-535; Santa Cruz, Dalas, TX, USA) or p-p38 (D3F9; Cell Signaling), and gently rocked at 4 °C overnight, followed by three 10 min washes in TBS containing 0,3% Tween-20. β-Actin (sc-47778, Santa Cruz) was used as a protein-loading control.
Techniques: Fluorescence, Western Blot, Flow Cytometry