BDNF-induced phospho-ERK signals and co-localization of phospho-ERK and TNFR1 after TNF-α treatment in DRG cultures. Phospho-ERK signals were detected after 5 nM TNF-α pre-treatment for 48 hours followed by 400 ng/ml BDNF for 0-40 min. Phospho-ERK and TNFR1 proteins were detected by double immunofluorescent stain. (A) Quantitative measurements of phospho-ERK fluorescent intensity in DRG cultures. Data are presented as fluorescent intensity of immunopositive neurons, expressed as mean ± SEM, and were analyzed using one-way ANOVA followed by Tukey test. **p < 0.01; compared to corresponding controls; #
p < 0.05, ###
p < 0.001; comparison between TNF-α and time-matched control groups (N = 9 per group). (B-G) After TNF-α treatment for 48 hours, an acute treatment with BDNF was applied for 10 min in cultured DRG neurons. Phospho-ERK is shown by green fluorescence and TNFR1 is shown by red fluorescence. (B-D) Photomicrographs illustrate the immunoreactivity of phospho-ERK (B) and TNFR1 (C) in DRG cultures of the control group (no TNF-α pre-treatment). (D) Merged image of (B) and (C). (E-G) Photomicrographs illustrate the immunoreactivity of phospho-ERK (E) and TNFR1 (F) in DRG cultures of the TNF-α group. (G) Merged image of (E) and (F). Inserts illustrate the phospho-ERK and TNFR1 signals that co-localized in a DRG neuron. Wide arrow, co-localized phospho-ERK and TNFR1; narrow arrow, TNFR1; arrowhead, phospho-ERK. Scale bars, 50 μm.