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1.
Figure 6

Figure 6. Functional Consequences of Restoration of KCNQ4 Surface Expression.. From: Distinct Roles of Molecular Chaperones HSP90? and HSP90? in the Biogenesis of KCNQ4 Channels.

A-K. Representative raw traces Whole-cell currents were recorded from transfected HEK293T cells. A. 0.5 µg WT KCNQ4 + 2.5 µg vector; B. 0.5 µg L274H + 2.5 µg vector; C. 0.5 µg L274H + 2.5 µg HSP90β; D. 0.25 µg L274H + 0.25 µg WT KCNQ4 + 2.5 µg vector; E. 0.25 µg L274H + 0.25 µg WT KCNQ4 + 2.5 µg HSP90β; F. 0.5 µg WT KCNQ4 + 2.5 µg HSP90β; G. 0.5 µg W276S + 2.5 µg vector; H. 0.5 µg W276S + 2.5 µg HSP90β; I. 0.25 µg W276S + 0.25 µg WT KCNQ4 + 2.5 µg vector; J. 0.25 µg W276S + 0.25 µg WT KCNQ4 + 2.5 µg HSP90β; K. non-transfected cells. L.Average whole-cell current densities (pA/pF) were calculated as the maximal current (pA) divided by the cell capacitance (pF). Each data point in the bar graph represents the average ± SEM of the indicated number of cells and was compared to the average current density of the WT channel (* p≤0.05, ** p≤0.01). The time scale on K. applies to all panels.

Yanhong Gao, et al. PLoS One. 2013;8(2):e57282.
2.
Figure 4

Figure 4. Effects of HSP40, HSP70, and HSP90β on KCNQ4 Surface Expression.. From: Distinct Roles of Molecular Chaperones HSP90? and HSP90? in the Biogenesis of KCNQ4 Channels.

A. B. & C. Representative Western bolt HEK293T cells were transfected with indicated combination of plasmid DNAs (0.4 µg of a KCNQ4 channel and 1 µg of a chaperone or a vector as indicated on the top of the blots). 24 hrs post transfection, KCNQ4 channels on cell surface were labeled with a mouse monoclonal antibody against the HA tag in KCNQ4 channels prior to cell lysis, then, isolated using Dynabeads Protein G (IP) and assessed by Western blot (IB). D. Summary data Each data point in the bar graphs represents the mean ±SD of three experiments (* p≤0.05, ** p≤0.01). Compared with HSP40 and HSP70, HSP90β showed the highest rescue efficiency of KCNQ4 mutants.

Yanhong Gao, et al. PLoS One. 2013;8(2):e57282.
3.
Figure 5

Figure 5. Rescue KCNQ4 Surface Expression in Cells Mimicking the Heterozygous Condition of DFNA2 Patients.. From: Distinct Roles of Molecular Chaperones HSP90? and HSP90? in the Biogenesis of KCNQ4 Channels.

A. & B. Representative Western blot HEK293T cells were transfected with WT and a mutant KCNQ4 channel at a ratio 1:1 plus various amount of HSP90β as indicated. 24 hrs post transfection, KCNQ4 channels on cell surface were labeled with a mouse monoclonal antibody against the HA tag in the first extracellular loop of KCNQ4 channels prior to cell lysis; then isolated using Dynabeads Protein G and analyzed by Western blot. C. & D. Summary data Each data point in the bar graphs represents the mean ±SD of three experiments (* p≤0.05, ** p≤0.01). Surface expression in cells co-expressing WT/L274H or WT/W276S could be restored to the level of WT KCNQ4 by over-expression of HSP90β.

Yanhong Gao, et al. PLoS One. 2013;8(2):e57282.
4.
Figure 3

Figure 3. Roles of Cochaperones HOP and CHIP in KCNQ4 Biosynthesis.. From: Distinct Roles of Molecular Chaperones HSP90? and HSP90? in the Biogenesis of KCNQ4 Channels.

A. & B. Co-immunoprecipitation of KCNQ4 channels with cochaperones HOP and CHIP HEK293T cells were transfected with 0.4 µg of pCMV6-XL5-HA-KCNQ4 (+) or pCMV6-XL5 (-) and cultured for 24 hrs. Immunoprecipitation (IP) was done using antibodies either against the HA tag in KCNQ4 channels or a cochaperone (HOP or CHIP). The precipitate was analyzed by Western blot (IB) using the indicated antibodies. Cell lysates were tested in parallel for HA-KCNQ4, cochaperones, and GAPDH as input controls. Association of HOP and CHIP with KCNQ4 channels were confirmed by co-precipitation of these proteins. C. & D. Effects of over-expression of HOP or CHIP on total KCNQ4 level HEK293T cells were transfected with 0.4 ug of pCMV6-XL5-HA-KCNQ4 (+) and different amount of cochaperone cDNA as indicated. 24 hrs post transfection, the cells were lysed in NP40 lysis buffer and the cell lysates were tested for total KCNQ4 proteins by Western blot. Over-expression of cochaperone HOP led to a significant increase, whereas over-expression of CHIP resulted in a marked decrease in total KCNQ4 level. E. & F. Effects of siRNA knockdown of HOP or CHIP on total KCNQ4 level HEK293T cells were transfected with 0.4 µg of pCMV6-XL5-HA-KCNQ4 (+) and various amount of siRNA as indicated. The transfected cells were cultured for 48 hrs and the cell lysates were tested for KCNQ4 proteins. Knockdown of cochaperone HOP led to a marked decrease, while knockdown of CHIP resulted in a significant increase in total KCNQ4 level. Each data point in the bar graphs represents the mean ±SD of three experiments (* p≤0.05, ** p≤0.01).

Yanhong Gao, et al. PLoS One. 2013;8(2):e57282.
5.
Figure 2

Figure 2. Roles of HSC70 and Cofactor DjA1 in KCNQ4 Biogenesis.. From: Distinct Roles of Molecular Chaperones HSP90? and HSP90? in the Biogenesis of KCNQ4 Channels.

A. & B. Co-immunoprecipitation of KCNQ4 channels with HSP70 or DJA1 HEK293T cells were transfected with 0.4 µg of pCMV6-XL5-HA-KCNQ4 (+) or pCMV6-XL5 (-) and cultured for 24 hrs. Immunoprecipitation (IP) was carried out respectively using a mouse monoclonal antibody against the HA tag in KCNQ4 channels or an antibody against HSC70 (or DJA1). Precipitated proteins were detected by Western blot (IB) using the indicated antibodies. Cell lysates were also tested for HA-KCNQ4, HSC70, and GAPDH as input controls. Both HSP70 and DJA1 were precipitated with KCNQ4 channels, and vice versa. C. & D. Effects of over-expression of HSC70 or DjA1 on total KCNQ4 level HEK293T cells were transfected with 0.4 µg of pCMV6-XL5-HA-KCNQ4 (+) and various amount of each chaperone cDNA as indicated. The transfected cells were cultured for 24 hrs and KCNQ4 proteins in the cell lysates were assessed by Western blot. Over-expression of either HSC70 or DJA1 induced a dramatic increase in total KCNQ4 level. E. & F. Effects of siRNA knockdown of HSC70 or DjA1 on total KCNQ4 level HEK293T cells were transfected with 0.4 µg of pCMV6-XL5-HA-KCNQ4 (+) and various amount of siRNA specifically targeting HSC70 or DjA1 as indicated. Non-specific siRNA (Ctrl.) were transfected in parallel as negative controls. 48 hrs post transfection, the cell lysates were collected and analyzed for total KCNQ4 proteins. Knockdown of HSC70 or DJA1 resulted in a significant decrease in total KCNQ4 level. Each data point in the bar graphs represents the mean ±SD of three experiments (* p≤0.05, ** p≤0.01).

Yanhong Gao, et al. PLoS One. 2013;8(2):e57282.
6.
Figure 1

Figure 1. Opposite Roles of Molecular Chaperones HSP90α and HSP90β in KCNQ4 Biogenesis.. From: Distinct Roles of Molecular Chaperones HSP90? and HSP90? in the Biogenesis of KCNQ4 Channels.

A. & B. Co-immunoprecipitation of KCNQ4 channels with the molecular chaperones HSP90 & HSP90β HEK293T cells were transfected with 0.4 µg of pCMV6-XL5-HA-KCNQ4 (+) or pCMV6-XL5 alone (-) and harvested 24 hrs post transfection. Immunoprecipitation (IP) was carried out respectively using a mouse monoclonal antibody against the HA tag in the first extracellular loop in KCNQ4 channels or an antibody against HSP90α (or HSP90β). Precipitated proteins were then analyzed by Western blot (IB) using the indicated antibodies; cell lysates were tested in parallel as input controls. Both HSP90α and HSP90β were co-immunoprecipitation with the KCNQ4 channels and vice versa, indicating that these two chaperones are associated with KCNQ4 channels. C. & D. Effects of over-expression of HSP90α or HSP90β on total KCNQ4 level HEK293T cells were transfected with 0.4 µg of pCMV6-XL5-HA-KCNQ4 (+) and various amount of HSP90α or HSP90β cDNA as indicated. The cells were then lysed in NP40 lysis buffer 24 hrs post transfection and the total amount of KCNQ4 proteins in the cell lysates were assessed by Western blot. Over-expression of HSP90β resulted in a significant increase, while over-expression of HSP90α led to a marked decrease in total KCNQ4 level. E. & F. Effects of siRNA knockdown of HSP90α or HSP90β on total KCNQ4 level HEK293T cells were transfected with 0.4 µg of pCMV6-XL5-HA-KCNQ4 cDNA (+) and various amount of siRNA specifically targeting HSP90α or HSP90β as indicated. Non-specific siRNA (Ctrl.) were used in parallel as negative controls. Total KCNQ4 proteins were assessed by Western blot 48 hrs post transfection. Specific knockdown of HSP90β led to a dramatic decrease, whereas knockdown of HSP90α resulted in a marked increase in total KCNQ4 level. Each data point in the bar graphs represents the mean ±SD of three experiments (* p≤0.05, ** p≤0.01).

Yanhong Gao, et al. PLoS One. 2013;8(2):e57282.

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