kcnt2  (Alomone Labs)

Journal: Aging (Albany NY)

Article Title: The local translation of KNa in dendritic projections of auditory neurons and the roles of KNa in the transition from hidden to overt hearing loss

doi: 10.18632/aging.102553

Figure Lengend Snippet: sm-FISH and immunocytochemistry localize transcripts and proteins for K Na 1.1 and K Na 1.2 in axons and cell bodies of spiral ganglion neurons (SGNs). Expression of K Na 1-encoding transcripts in the SGNs was examined using smFISH and standard immunocytochemistry in the organ of Corti (OC)/SGN preparations from 1-mo old C57 mice ( B – I ). ( A ) Schematic illustration of the inner hair cell (IHC), type I SGN, the peripheral axon, and cell body. The unmyelinated terminal, heminode, and nodes of Ranvier are noted, but not to scale. ( B ) RNA molecules encoding for K Na 1.1 ( Kcnt1 ), and ( C ) K Na 1.2 ( Kcnt2 ), were detected as fluorescent spots (purple, arrow) in TUJ1-positive (green) SGN axons, IHCs were labeled with myosin 7A antibody (red), and merged images are shown. Axonal Kcnt1 mRNA were prominent, but only scant Kcnt2 mRNA spots were detected compared to the double knockout (DKO) samples ( J ). Scale bar = 10 μm ( D – E ) Images of cochlear sections of 1-mo old mice show that K Na 1.1 (red) protein is expressed in the auditory nerve in D. Consistent with the faint expression of Kcnt2 mRNA in the axons in ( E ) there was virtually little or no detectable expression of K Na 1.2 in axons of the auditory nerve. Scale bar = 10 μm. ( F – G ) mRNA spots (purple spots) encoding K Na 1.1 ( Kcnt1 ), and K Na 1.2 ( Kcnt2 ) in the cell bodies of SGNs. Very few spots for Kcnt2 mRNA were detected. Sections were co-labeled with neuronal (TuJ1, green) and nuclei markers (4,6-diamidino-2-phenylindole, DAPI, blue) Scale bar = 5 μm. ( H – I ) Images of the SGNs show K Na 1.1 (red) protein is expressed in cell bodies of the auditory nerve. In keeping with low levels of expression of mRNA, K Na 1.2 protein expression was faintly positive. The mean number of RNA molecules detected per SGN was calculated as described in the Methods. Kcnt1 levels were higher compared to Kcnt2 in both mRNA and protein levels. ( J ) (Upper panel). Photomicrograph showing SGN mRNA spots (red spots) encoding Gapdh (data was obtained from DKO tissue) . (Lower panel) DKO cochlear section, using kcnt1 probe serving as negative controls. Similar data were obtained using the kcnt2 probe (data not shown). Scale bar = 5 μm. ( K ) Values of mRNA spots in axons and cell bodies were normalized against Gapdh mRNA spots/100 μm 2 (11 ± 2 spots (n = 31)) are summarized in the form of bar graphs. The mean (mean ± SD) was (cell body, kcnt1 = 0.38 ±.0.11; kcnt2 = 0.16 ± 0.06; DKO = 0.02 ± 0.04; n = 11 animals; derived from 50 randomly selected cells and evaluated by 5 blinded individuals. The mean (mean ± SD) was (axons, kcnt1 = 0.22 ±.0.07; kcnt2 = 0.08 ± 0.06; DKO = 0.03 ± 0.05; n = 11 animals; derived from 50 randomly selected cells and evaluated by 5 blinded individuals.

Article Snippet: The following primary antibodies were used: rabbit anti-KCNT1, KCNT2 (Alomone, Jerusalem, Israel), rabbit anti-cleaved caspase-3 and -9 (Cell Signaling Technology, Danvers, MA), rabbit and mouse anti-myosin 7a (Proteus Biosciences, Novus Biological and Developmental Studies Hybridoma Bank (DSHB)) mouse anti-PSD95 (Millipore), mouse anti-CtBP2 (BD Biosciences, San Jose, CA), rabbit anti-active-caspase-9 and rabbit anti-active-caspase-3 (Abcam, Cambridge, MA), rabbit anti-N-IP3 R1 (Biorbyt, San Francisco, CA) rabbit anti-pIP3 R1 (Invitrogen, Carlsbad, CA), rabbit anti-IP3 R1 (cleavage site, Novus Biologicals, Centennial, CO), rabbit anti-ER lumen IP3R1 (Sigma), rabbit anti-calnexin (ER marker, Abcam) and mouse anti-neuronal class III ß-Tubulin (TUJ1) (Covance, Princeton, NJ).

Techniques: Fluorescence In Situ Hybridization, Immunocytochemistry, Expressing, Mouse Assay, Immunohistochemistry, Labeling, Double Knockout, Derivative Assay