NeNaC2 Protein Detection by Western Blot

For NeNaC2 Western blot, we used the method as previously described in ref. ^{82 (link)}. In brief, custom polyclonal antibodies raised against recombinant fragment antigens generated by immunization of rabbits (GenScript, USA). The sequence of the recombinant fragment was ITGCLSLYDLKLIAAVMSCPVAQRHFETEEDKKDEDEDDRAEDPVDENPDDTITVSQMWQDFLHTLTLHGFRFVFERGPTHHHHHH. Equal amounts of protein were run on 4–15% Mini-PROTEAN® TGX™ Precast Protein Gel (Bio-Rad, USA) followed by blotting to a Polyvinylidene fluoride (PVDF) membrane (Bio-Rad). Membrane was incubated overnight with polyclonal antibody against NeNaC2 or monoclonal mouse anti-GAPDH (Abcam, UK) with a dilution of 1:1000 at 4 °C overnight, then washed and incubated for 1 h with peroxidase-conjugated anti-mouse or anti-rabbit antibody (Jackson ImmunoResearch, USA) with a dilution of 1:10,000. Detection was performed with the Clarity™ Max ECL kit (Bio-Rad) according to the manufacturer’s instructions and visualized with a CCD camera of the Odyssey Fc imaging system (Li-COR Biociences, USA).