Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression in Müller cells suppress the MYD88/IRAK1/TRAF6/NF-κB p65 inflammatory signaling pathway. (A) Representative immunoblots showing changes in MYD88, IRAK1, TRAF6, and NF-κB p65 expression levels in microglia co-cultured with normal or activated Müller cells infected with LV-NC, Kir4.1, and Kir4.1 Tyr 9 Asp lentiviruses. (B, D, F, H) Bar charts summarizing the average densitometric quantification of immunoreactive bands for MYD88 (B), IRAK1 (D), TRAF6 (F), and NF-κB p65 (H) in microglia co-cultured with normal or activated Müller cells. n = 5 for each group. * P < 0.05, ** P < 0.01, vs. the + normal Müller cells group. Unpaired two-tailed t -test. (C, E, G, I) Bar charts summarizing the average densitometric quantification of immunoreactive bands for MYD88 (C), IRAK1 (E), TRAF6 (G), and NF-κB p65 (I) in microglia co-cultured with normal or activated Müller cells. n = 5 for each group. * P < 0.05, ** P < 0.01, vs. LV-NC + normal Müller cells group. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed. eGFP: Enhanced green fluorescent protein; IRAK1: IL-1 receptor associated kinase 1; LV-NC: eGFP control lentiviruses; MYD88: myeloid differentiation primary response protein 88; NF-κB P65: nuclear factor kappa B P65; TRAF6: TNF receptor associated factor 6.

Article Snippet: The primary antibodies used in this study included: mouse anti-GFAP (1:500, Sigma-Aldrich, St. Louis, MO, USA, Cat# G6171, RRID: AB_1840893), rabbit anti-Kir4.1 (1:400, Alomone, Jerusalem, Israel, Cat# APC-035, RRID: AB_2040120; APC-165 for membrane protein, RRID: AB_2041043), anti-B cell lymphoma/leukemia-2 (Bcl-2; 1:1000, Abclonal Biotechnology Co., Ltd, Wuhan, Hubei, China, Cat# A0208, RRID: AB_2757022), rabbit anti-Bcl-2-associated X protein (Bax; 1:1000, Abcam, Cambridge, MA, USA, Cat# ab32503, RRID: AB_725631), anti-myeloid differentiation primary response protein 88 (MYD88; 1:1000, Novus Biologicals, Littleton, CO, USA, Cat# NB100-56698SS, RRID: AB_838599), anti-nuclear factor kappa B p65 (NF-κB p65; 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3033T, RRID: AB_331284), goat anti-translocator protein (TSPO; 1:1000, Novus Biologicals, Cat# NB100-41398, RRID: AB_788260), rabbit anti-cleaved caspase 3 (1:500, Zen-Bioscience, Chengdu, Sichuan, China, Cat# 341034, RRID: AB_2924432), rabbit anti-IL-1 receptor-associated kinase 1 (IRAK1; 1:1000, Abcam, Cat# ab180747, RRID: AB_2895218), rabbit anti-TNF receptor-associated factor 6 (TRAF6; 1:1000, Abcam, Cat# ab33915, RRID: AB_778572), mouse anti-β-actin (1:10,000, Sigma-Aldrich, Cat# A5441, RRID: AB_476744), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000, Sigma-Aldrich, Cat# G9545, RRID: AB_796208), and rabbit anti-Na-K ATPase (1:8000, Abcam, Cat# ab167390, RRID: AB_2890241).

Techniques: Over Expression, Western Blot, Expressing, Cell Culture, Infection, Two Tailed Test, Control