Sc 57954

Recombinant human TGF-β1 and TGF-β1 receptor inhibitor (SB431542) were purchased from R&D Systems. Carbon particles were obtained from Mitsubishi Chemical Corporation. Bleomycin was obtained from Tianjin Taihe Pharmaceutical Co., Ltd. Abs used were as follows: anti-SRSF6 (PA5-41810, Invitrogen and sc-57954, Santa Cruz Biotechnology), anti-COL1A2 (14695-1-AP, Proteintech), anti-ACTA2 (14395-1-AP, Proteintech), anti-SOX4 (B-7) (sc-518016, Santa Cruz Biotechnology), anti-WNT5A (abs113167, Absin), anti-TGF-β1 (21898-1-AP, Proteintech), anti-SMAD5 (12167-1-AP, Proteintech), anti-phospho-SMAD1/5/9 (P-SMAD1/5/9) (13820, Cell Signaling Technology), anti-SMAD2/3 (8685, Cell Signaling Technology), anti-phospho-SMAD2/3 (p-SMAD2/3) (8828, Cell Signaling Technology), anti-GFP (2956, Cell Signaling Technology), anti-SRSF5 (16237-1-AP, Proteintech), anti-GAPDH (60004-1, Proteintech), and IgG (10283-1-AP, Proteintech).

IMCD, mouse cortical collecting duct principal cell line mpkCCD cells or renal tissue were lysed in protein lysis buffer for 15 mins on ice before protein was extracted. Immunoblotting was performed with primary antibodies against aquaporin-2 (AQP2, 1:3000), aquaporin-3 (AQP3 1:1000) 35 (link), p-Ser256 AQP2 (1:1000, ab111346 Abcam, USA), NLRP3 (1:1000, ab214185 Abcam, USA; 1:1000), Caspase-1 (p20) (1:1000, AB1871 Milipore, USA), ASC (1:1000, sc-514414 Santa Cruz Biotechnology, USA), IL-1β (1:1000, sc-57954 Santa Cruz Biotechnology, USA), SQSTM1 (1:1000, 5114 Cell signaling technology, USA), LC3B (1:1000 2775S Cell signaling technology, USA) followed by the addition of horseradish peroxidase-labelled secondary antibodies. The blots were visualized with ECL detection systems. Densitometric analysis was performed using AlphaEase Software.
The samples subjected to IP assay were incubated with an anti-ASC antibody 1:10 (sc-514414 Santa Cruz Biotechnology, USA) in IP buffer overnight at 4 °C. Protein A-sepharose beads were added to the samples, which incubated for another 12 h. The sample were then washed and resuspended, and Western blotting was performed as described previously.