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. 2017 May 15;6:e24318. doi: 10.7554/eLife.24318

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© 2017, Cunningham et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 8. — (A–E) HEK293 cells were transfected with the constructs indicated on top of each panel to perform co-immunoprecipitation (CoIP) experiments. Immunoprecipitations were carried out with HA (A,B,E) or Flag (C,D) antibodies, followed by Western blotting to detect proteins. The upper panels show CoIP results, the middle row shows IP results and the lower rows show input protein. White asterisks indicate 25 kDa light-chain IgG bands from antibodies used for IP. (F) P6 whole mount cochleas from Tomt^add/+ (left panels) and Tomt^add/add mice (right panels) stained with anti-TMHS/LHFPL5 (Xiong et al., 2012) and phalloidin. Scale bar in (F) = 5 µm, applies to both panels. (G) OHCs from P3 wild-type (C57BL/6J) (left) and Tomt^add/add (right) mice were injectoporated with TMIE-HA, cultured for 24 hr, fixed and stained with anti-HA antibody and phalloidin. TMIE-HA localizes to the hair bundle of both wildtype and Tomt^add/add OHCs. (H) Outer hair cells from P3 Tomt^del/+ (left) and Tomt^del/del (right) mice were injectoporated with MYC-TMC1, cultured for 24 hr, fixed and stained with anti-MYC antibody and phalloidin. MYC-TMC1 localizes to the hair bundle of Tomt^del/+ but is absent from the hair bundle of Tomt^del/del OHCs. Optical sections at the level of the hair bundle and cell body are shown. (I) Outer hair cells from P3 Tomt^del/+ (left) and Tomt^del/del (right) mice were injectoporated with MYC-TMC2, cultured for 24 hr, fixed and stained with anti-MYC antibody and phalloidin. MYC-TMC2 localizes to the hair bundle of Tomt^del/+ but is absent from the hair bundle of Tomt^del/del OHCs. Optical sections at the level of the hair bundle and cell body are shown. (J) Outer hair cells from P3 Tomt^del/del mice were injectoporated with mTOMT-GFP and MYC-TMC2, cultured for 24 hr, fixed and stained with anti-MYC antibody and phalloidin. MYC-TMC2 localizes to the hair bundle of Tomt ^del/del hair cells in the presence of exogenous mTOMT-GFP. Optical sections at the level of the hair bundle and cell body are shown. (K) Quantification of injectoporation experiments shown in (H–J). The data are plotted as the proportion of injectoporated outer hair cells for each genotype and construct that exhibited TMC protein localized to stereocilia. The proportion of cells for each condition are indicated on the columns. Only healthy transfected hair cells, as determined by stereocilia morphology, were included for analysis. Scale bar in (G) = 5 µm, applies to (G–J).

DOI: http://dx.doi.org/10.7554/eLife.24318.014

Figure 8—figure supplement 1. — (A–E) HEK293 cells were transfected with the constructs indicated on top of each panel to perform co-immunoprecipitation (CoIP) experiments. Immunoprecipitations were carried out with HA (A,B,E) or Flag (C,D) antibodies, followed by Western blotting to detect proteins. The upper panels show CoIP results, the middle row shows IP results and the lower rows show input protein. White asterisks indicate 25 kDa light-chain IgG bands from antibodies used for IP. (F) P6 whole mount cochleas from Tomt^add/+ (left panels) and Tomt^add/add mice (right panels) stained with anti-TMHS/LHFPL5 (Xiong et al., 2012) and phalloidin. Scale bar in (F) = 5 µm, applies to both panels. (G) OHCs from P3 wild-type (C57BL/6J) (left) and Tomt^add/add (right) mice were injectoporated with TMIE-HA, cultured for 24 hr, fixed and stained with anti-HA antibody and phalloidin. TMIE-HA localizes to the hair bundle of both wildtype and Tomt^add/add OHCs. (H) Outer hair cells from P3 Tomt^del/+ (left) and Tomt^del/del (right) mice were injectoporated with MYC-TMC1, cultured for 24 hr, fixed and stained with anti-MYC antibody and phalloidin. MYC-TMC1 localizes to the hair bundle of Tomt^del/+ but is absent from the hair bundle of Tomt^del/del OHCs. Optical sections at the level of the hair bundle and cell body are shown. (I) Outer hair cells from P3 Tomt^del/+ (left) and Tomt^del/del (right) mice were injectoporated with MYC-TMC2, cultured for 24 hr, fixed and stained with anti-MYC antibody and phalloidin. MYC-TMC2 localizes to the hair bundle of Tomt^del/+ but is absent from the hair bundle of Tomt^del/del OHCs. Optical sections at the level of the hair bundle and cell body are shown. (J) Outer hair cells from P3 Tomt^del/del mice were injectoporated with mTOMT-GFP and MYC-TMC2, cultured for 24 hr, fixed and stained with anti-MYC antibody and phalloidin. MYC-TMC2 localizes to the hair bundle of Tomt ^del/del hair cells in the presence of exogenous mTOMT-GFP. Optical sections at the level of the hair bundle and cell body are shown. (K) Quantification of injectoporation experiments shown in (H–J). The data are plotted as the proportion of injectoporated outer hair cells for each genotype and construct that exhibited TMC protein localized to stereocilia. The proportion of cells for each condition are indicated on the columns. Only healthy transfected hair cells, as determined by stereocilia morphology, were included for analysis. Scale bar in (G) = 5 µm, applies to (G–J).

DOI: http://dx.doi.org/10.7554/eLife.24318.014