Immunoblotting

Tissue homogenates were prepared as described by Yang et al. (2016a). After homogenization and centrifugation, renal supernatants were collected and processed immediately to determine protein concentration or stored at −80°C for immunoblotting. Protein concentrations were identified using the Protein Assay Kit (Bio-Rad, Hercules, CA, USA), with BSA (Sigma) as a standard.

Immunoblotting was performed as previously described (Wang et al., 2008; Yang et al., 2011) with some modifications. Briefly, renal lysates were heated at 100°C for 5 min or at 37°C for 30 min (for TnFXYD8 or NKA, respectively), followed by 15% (for TnFXYD8) or 7.5% (for NKA) SDS-PAGE. Following electrophoresis, proteins were transferred to PVDF membranes (Millipore), and the blots were pre-incubated at room temperature for 1 h in PBST (PBS with 0.05% Tween 20) containing 5% (w/v) non-fat dried milk to minimize non-specific binding. Then, the blots were incubated at room temperature for 1 h with either TnFXYD8 (200,000 × dilution), NKA (2,500 × dilution), or actin (5,000 × dilution) in 5% (w/v) non-fat dried milk and 0.05% sodium azide (Sigma) in PBST, followed by incubation at room temperature for 1 h with a secondary antibody diluted in PBST. Blots were developed with an ECL kit (#34082; Pierce). Immunoblots were photographed and imported as JPEG files into the image analysis software package (MCID 7.0; Imaging Research, Ontario, Canada). Results were converted to numerical values to permit comparison of immunoreactive band intensities. The renal homogenates from FW-acclimated pufferfish were used as the internal control among different immunoblots (data not shown). The intensity of the immunoreactive band of the internal control in the immunoblot was set to one and used as a standard for normalizing relative intensities of other bands in the immunoblots. Relative abundance of target proteins was calculated using the following formula (TnFXYD8_n/actin_n)/(TnFXYD8_{internal control}/actin_{internal control}) (Chang et al., 2016a,b).