Em1101

The mCherry and Flag fusion constructs were verified by DNA sequencing and transformed in pairs into PH-1. Transformants expressing the fusion constructs were confirmed by western blot analysis. In addition, the transformants bearing a single fusion construct were used as references. For Co-IP assays, total proteins were extracted and incubated with the anti-Flag (Abmart, Shanghai, China) agarose overnight at 4°C as described previously [97 (link)]. Proteins eluted from agarose were analyzed by western blotting detection with the monoclonal anti-mCherry ab125096 (Abcam, Cambridge, UK) antibody. The protein samples were also detected with monoclonal anti-GAPDH antibody EM1101 (Hangzhou HuaAn Biotechnology co., Ltd.) as a reference.

For western blotting assay, protein samples of strains were prepared and extracted as described previously [96 (link)]. Proteins separated on the SDS-PAGE gel were transferred onto a polyvinylidene fluoride membrane with a Bio-Rad electroblotting apparatus. The polyclonal anti-Flag A9044 (Sigma, St. Louis, MO, USA), monoclonal anti-GFP ab32146 (Abcam, Cambridge, MA, USA) and monoclonal anti-mCherry ab125096 (Abcam, Cambridge, UK) antibodies were used at a 1:2000 to 1:10000 dilution for immunoblot assays. The samples were also detected with the monoclonal anti-GAPDH antibody EM1101 (Hangzhou HuaAn Biotechnology co., Ltd.) as a reference. Incubation with a secondary antibody and chemiluminescent detection were performed as described previously [97 (link)]. The experiment was repeated three times independently.

Total protein from the kidney tissues was isolated after being lysed by RIPA buffer (01408, Beyotime Biotechnology, China) containing PMSF, protease, and protein phosphatase inhibitor for 30 min on ice. All samples were centrifuged at 12000 g for 15 min (4°C). Protein concentration was determined using the BCA assay kit (P0012, Beyotime Biotechnology, China). An equal amount of protein (40 μg) was subjected to 8–10% SDS-PAGE gel and then transferred to PVDF membranes (162–0177, BIO-RAD, USA). The membranes were blocked with 5% BSA and incubated overnight with the following antibodies at 4°C: antinephrin (ab216341, Abcam, Cambridge, UK), anti-Wilms Tumor 1 (WT1) (ab89901, Abcam, Cambridge, UK), anti-Aquaporin 1 (ab168387, Abcam, Cambridge, UK), and anti-GAPDH (EM1101, Huaan Biotechnology, Hangzhou, China). After incubating with peroxidase-conjugated secondary antibodies (goat anti-rabbit-antibody, LI-COR Biosciences, USA), the protein expression bands were developed with chemiluminescence. The densitometry of the brands was then calculated using the Odyssey near-infrared dual-color laser imaging system (Odyssey Clx, LI-COR Biosciences, USA).