Figure 3
Expression of SCN10A mRNA and NaV1.8 α subunit protein in human kidney tissues. (A) Detection of SCN10A mRNA from human kidney tissues and HEK293T by RT-PCR method. SCN10A mRNA extracted from sperm was used as a positive control. A region of SCN10A mRNA covering exons 2–3 was analyzed, and mRNA of a house-keeping gene– ACTB was used as an internal control. (B) Western-blot analysis showed NaV1.8 α subunit of voltage-gated sodium channel expression in four human kidney tissue samples. Actin was used as loading control. (C) Detection of NaV1.8 α subunit protein by immunohistochemistry. The protein was stained in proximal tubules (2 upper panels in the first column) as well as collecting duct (2 lower panels in the first column) of human kidney. AQP1 (second column) was used as protein marker of proximal tubule and AQP2 (fourth column) as protein marker of collecting duct. Rabbit antibody was used as isotype control (third column). Hematoxyline and eosin staining of kidney tissue is shown in the fifth column. The original magnification was 40×. (D) Detection of NaV1.8 α subunit protein by double immunofluorescene staining in human kidney tissue. Fresh frozen human kidney-tissue section was incubated with mouse (Ms) or rabbit (Rb) antibody as isotype control (the first and second top panels), incubated with Hoechst 33258 for nuclear staining (blue), and incubated with mouse (Ms) anti-alpha 1 Na+/K+ ATPase antibody (red) for membrane staining (the first bottom panel) or rabbit (Rb) anti-NaV1.8 antibody (green) for NaV1.8 α subunit protein staining (the second bottom panel). The first and second bottom panels were merged in the third bottom panel and then enlarged. The original magnification was 20×. Images of the gels and blots cropped from different parts of the same gel/blot, or from different gels/blots were separated by white space. The full-length gels and blots are presented in Supplementary Fig. S9.
