Protoblue safe

CtpA and LbcA were purified as described above. To purify His₆-PA1198, E. coli strain M15 (pREP4) (Qiagen) containing pAJD2948 was grown in 500 mL of LB broth at 37°C to an OD₆₀₀ of 0.6 to 1.0. Protein production was induced with 1 mM IPTG at 37°C for 3 h. His₆-PA1198 was purified natively by Ni-NTA-agarose affinity chromatography in 50 mM NaH₂PO₄ and 300 mM NaCl, as recommended by the manufacturer (Qiagen). Protein was eluted in 50 mM NaH₂PO₄, 300 mM NaCl, 50 mM imidazole, pH 8. Assays were done as described previously (23 (link), 36 (link)). The samples were separated by SDS-PAGE and stained with ProtoBlue Safe (National Diagnostics). For mutant CtpA protein experiments, ImageJ analysis was used to determine the densities of the His₆-PA1198 bands. His₆-PA1198 degradation was quantified by comparing His₆-PA1198 band density to that in the reaction with inactive CtpA-S302A. Averages from two independent experiments are reported.

Rats were placed for 16 h in metabolic cages with ad libitum access to food and drinking water. The body weight, water intake and urinary volume were measured, and urine was collected over ice. Urine and blood parameters were measured by using a UniCel DxC 800 Pro Synchron (Beckman Coulter, Fullerton, CA, USA). The concentration of the LMW Clara cell protein (CC16) in urine was measured in duplicate by enzyme-linked immunosorbent assay (ELISA; abx155347, ABEXA, Cambridge, UK). Albuminuria was measured via Coomassie Blue staining by using ProtoBlue Safe (EC-722, National Diagnostics, Atlanta, GA, USA) according to the manufacturer’s instructions.

The purified prostasin-HAI-1 complexes from the immunoaffinity column were resolved by a 7.5% SDS-PAGE under non-boiled conditions. A gel strip was then sliced from the gel from the previous step, which was then boiled with SDS sample buffer containing no reducing agent. The boiled gel strip was placed on the top of a new 7.5% SDS-PAGE gel and subjected to electrophoresis in the second dimension. The first and second dimensional gels were stained using Protoblue Safe (National Diagnostics). The protein spots of interest were sliced from the gel and subjected to proteomics-based protein identification provided as service by ProtTech (Eagleville, PA).