Roles of HSC70 and Cofactor DjA1 in KCNQ4 Biogenesis.

<p><i>A. & B. Co-immunoprecipitation of KCNQ4 channels with HSP70 or DJA1</i> 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. <i>C. & D. Effects of over-expression of HSC70 or DjA1 on total KCNQ4 level</i> 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. <i>E. & F. Effects of siRNA knockdown of HSC70 or DjA1 on total KCNQ4 level</i> 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).