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Fig. 9 | Acta Neuropathologica Communications

Fig. 9

From: EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

Fig. 9

Activation of ephrinB/EphB forward signaling induces translocation of NF-κB p65 from the cytoplasm into the nucleus. a, Confocal laser microphotographs of cultured Müller cells, stained with the antibody against NF-κB p65 (green), showing the changes in NF-κB p65 protein expression in IgG-Fc treatment (Ctr) and ephrinB-Fc-treatment for different periods of time (a1-a7). b1-b7 are DAPI images. c1-c7 are merged images. Scale bar: 10 μm, for all the images. b, Representative immunoblots showing the changes in NF-κB p65 protein levels in whole cell extracts obtained from IgG-Fc-treated cells (Ctr) and those treated with ephrinB1-Fc for different periods of time. c, Bar chart summarizing the average densitometric quantification of immunoreactive bands of NF-κB p65 in Müller cells treated with ephrinB1-Fc for different periods of time. n = 5 for all groups. ** P < 0.01 vs. Ctr. d, Representative immunoblots showing the changes of NF-κB p65 in nucleus component of Müller cells treated with IgG-Fc (Ctr), and with ephrinB1-Fc for different periods of time. e, Bar chart summarizing the average densitometric quantification of immunoreactive bands of nucleus NF-κB p65 under the conditions as shown in D. n = 6 for all groups. * P < 0.05, and *** P < 0.001 vs. Ctr. f, Representative immunoblots showing the changes in NF-κB p65 protein levels in the nucleus component of Müller cells obtained in the presence of ephrinB1-Fc, with or without in the presence of LY294002. LY294002 (2 μM/10 μM) was added to the medium 30 min before a 3 h ephrinB1-Fc treatment. g, Bar chart summarizing the average densitometric quantification of immunoreactive bands of nucleus NF-κB p65 under the conditions as shown in F. n = 4 for all groups. ** P < 0.01 vs. Ctr (IgG-Fc treated group). h, Summary data showing that pre-incubation with PDTC inhibited the ephrinB1-Fc treatment induced increase in TNF-α mRNA levels in Müller cells. PDTC (10 μM/50 μM) was added to the medium 30 min before the ephrinB1-Fc treatment. n = 4 for all groups. i, Summary data showing that PDTC blocked the ephrinB1-Fc treatment induced increase in TNF-α protein levels in Müller cells. PDTC (50 μM) was added to the medium 30 min before the ephrinB1-Fc treatment. n = 4 for all groups. ** P < 0.01 and *** P < 0.001 vs. Ctr (IgG-Fc treated group)

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