Journal: The Journal of Clinical Investigation

Article Title: A disease mutation reveals a role for Na V 1.9 in acute itch

doi: 10.1172/JCI122481

Figure Lengend Snippet: (A) After healing, wounds from scratching left marks that resembled bruising (black arrows) in the p.L811P patient. (B) Schematic diagram of the flexible accelerated Neo-STOP tetracycline-inducible (FAST) cassette illustrating the generation of global-knockout NaV1.9 mice (red box) and Cre-mediated expression of sfGFP-tagged NaV1.9 mice (green box). (C) A DRG section from an sfGFP-tagged NaV1.9 (top) and a WT mouse (bottom) showing the overlap between endogenous fluorescent signal (green) and autofluorescence, followed by staining with an antibody against GFP (red). (D) Western blot of DRG tissue from a WT, an sfGFP-NaV1.9, and an NaV1.9–/– mouse stained for GFP. An HSP90 antibody was used as a loading control. (E) Western blot of tissues taken from an sfGFP-NaV1.9 mouse and stained for GFP, stripped, and reprobed for NaV1.9 using a commercial antibody. An HSP90 antibody was used as a loading control. TG, trigeminal ganglia. (F and G) Representative current traces from ND7/23 cell lines expressing WT (black) or sfGFP-NaV1.9 (green) channels. (H–J) Current-voltage (I-V) (H) and deduced conductance-voltage (G-V) (I) and steady-state inactivation (SSI) (J) relationships of WT (black) and sfGFP-NaV1.9 (green). (G-V: WT-NaV1.9 V1/2 = –25.5 ± 0.5 mV, n = 16; GFP-NaV1.9 V1/2 = –24.0 ± 0.2 mV, n = 11, P = 0.52; SSI: WT-NaV1.9 V1/2 = –29.3 ± 3.5 mV, n = 7; GFP-NaV1.9 V1/2 = –26.6 ± 1.7 mV, n = 7, P = 0.49.). Data are represented as mean ± SEM. Scale bar: 50 μm.

Article Snippet: Primary antibodies used were rabbit anti-sfGFP (1:2,000; gift from Ramanujan Hegde), rabbit anti-Na V 1.9 (1:5,000; ASC-017, Alomone Labs), and rabbit anti-HSP90 (1:1,000; C45G5, Cell Signaling).

Techniques: Knock-Out, Expressing, Staining, Western Blot