Bca assay kit

Cells at about 90% confluence were collected in ice-cold phosphate buffered saline (PBS) and centrifuged at 1000 rpm at 4 °C for 5 min. Total protein was extracted from the stably transfected Hek293 cells by means of lysis buffer supplemented with protease inhibitors (Sangon Biotech, Shanghai, China). The protein concentration was estimated using a bicinchoninic acid (BCA) assay kit (Sangon Biotech, Shanghai, China). After incubation on ice for 30 min, the extracts were centrifuged at 14,000 rpm at 4 °C for 15 min, followed by resolving using 10% SDS-polyacrylamide gel electrophoresis and transferring to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The extracts were subjected then to immunoblotting with a 1:1000 dilution anti-Cx50 rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), as well as a 1:5000 dilution anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) at 4 °C, overnight. Fluorescent secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:5000 dilution was used for incubation. The blots were analyzed using a ChemiDoc^TM MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA), in which the expression levels of the target protein were normalized relative to β-actin expression.

The Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, United States). The BCA assay kit was purchased from Sangon Biotech (Shanghai, China). Dithiothreitol (DTT, 3483–12–3), 2,2,2-trifluoroethanol (TFE, 99%, 75–89–8), acetaldehyde-¹³C2 (¹³CH₃¹³CHO, 99 atom % 13C, 1632–98–0), acetaldehyde (CH₃CHO, 99%, 75–07–0), sodium cyanoborohydride (NaBH₃CN, 25,895–60–7), ammonia solution (NH₄OH, 7664–41–7), acetonitrile (ACN, 75–05–8), formic acid (FA, 64–18–6), trypsin, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Ultra-pure water was obtained on-site using the Millipore Simplicity System (Billerica, MA, United States). Ammonium bicarbonate (ABC) was purchased from Sangon Biotechnology (Shanghai, China). ZIC-HILIC particles were purchased from Thermo Fisher Scientific (Waltham, MA United States). PNGase F was purchased from New England Biolabs (Ipswich, MA, United States).

The protein extraction was performed according to Bo's et al. (2020 (link)) method with minor modifications. Frozen lotus embryos were mixed the steel beads and ground at the power of 60 Hz for 2 min. The samples were then supplemented with 1 ml of extraction buffer and mixed with Tris-phenol buffer for 30 min at 4°C. Next, the mixtures were centrifuged at 7,100 g for 10 min at 4°C and the phenol supernatants were collected. The supernatants were added to 5 times the volumes of 0.1 M cold ammonium acetate-methanol buffer and precipitated at −20°C overnight. After precipitation, the samples were centrifuged at 12,000 g for 10 min and the precipitation was collected. Then, the precipitation was washed by cold methanol and centrifuged at 12,000 g for 10 min at 4°C and the precipitation was collected. Methanol was then replaced by acetone and the wash stem was repeated twice to remove methanol contamination. Afterward, the samples were centrifuged at 12,000 g for 10 min at 4°C to collect precipitation and dried at room temperature for 3 min and then dissolved in lysis buffer for 3 h. Finally, the samples were centrifuged at 12,000 g for 10 min to collect supernatants. The supernatants were centrifuged again to remove precipitation completely. Protein concentration was determined by a BCA assay kit (Sangon Biotech, Shanghai, China) and aliquoted to store at −80°C.

The total protein of cells and rat kidney tissues was extracted with RIPA Lysis Buffer (C500007, Sangon, China), and the protein concentration was determined using BCA assay kit (C503021, Sangon, China). An equivalent of 30 μg protein extract and 5 µL marker (PR1910, Solarbio, China) were resolved on the SDS-PAGE, followed by being transferred onto PVDF membrane (IPFL00010, Millipore, USA). Next, the protein blot was blocked with skim milk, and probed with primary antibodies, followed by further incubation with appropriate secondary antibody goat anti-rabbit IgG (1:2,000, ab7090; abcam, UK). Subsequently, immunoreactive bands were detected using an ECL kit (ab133409; abcam). GAPDH served as an internal control. The protein bands on X-ray films were quantified with a Tanon 5200 imaging system (Tanon, China). The primary antibodies from abcam and Cell Signaling Technology (CST) are listed as follows: Bax (1:2,000; Rabbit; ab182733, 21 kDa; abcam), Bcl-2 (1:500; Rabbit; ab194583, 26 kDa; abcam), Cleaved Caspase 3 (1:1,000; Rabbit; #9661, 17 kDa; CST), p-Smad3 (1:2,000; Rabbit; ab52903, 48 kDa; abcam), Smad3 (1:1,000; Rabbit; ab40854, 48 kDa; abcam), p-protein kinase A (p-PKA, 1:1,000; Rabbit; #5661, 42 kDa; CST), PKA (1:1,000; Rabbit; #4782, 42 kDa; CST), NADPH oxidase 4 (Nox4, 1:1,000; Rabbit; ab154244, 67 kDa; abcam), and GAPDH (1:10,000; Rabbit; ab181602, 36 kDa).