Anti ire1α

Protein sample preparation and Western immunoblotting were performed as previously described (Qin et al., 2011 (link); Pandey et al., 2017 (link)). Primary antibodies used in the immunoblotting analysis included anti-p-ULK1, anti-ULK1, anti-ASK1, anti-p-ASK1, anti-SAPK/JNK, anti-p-SAPK/JNK (Cell signaling); anti-AMPKα, anti-p-AMPKα, anti-Atg9a (Thermo Scientific); anti-LC3, anti-p-ASK1 and anti-GAPDH (Santa Cruz Biotech., Inc); anti-BAK, anti-BAX (EMD Millipore), anti-p-IRE1α (GeneTex, Inc), anti-IRE1α (Novus Biologicals). Dilution of primary antibodies was 1:1,000. Secondary antibody HRP anti-IgG (Sigma-Aldrich, USA, 1:1,000~5,000) was used in the immunoblotting analysis. Densitometry of blots was performed using the ImageJ software package (http://rsbweb.nih.gov/ij/). The relative expression levels of target proteins and the ratio of blot LC3-II/LC3-I at the indicated time points were calculated as previously described (Qin et al., 2011 (link)). All Westerns were performed in triplicate and representative images are shown.

The kidney tissue sections were fixed with formalin and then embedded in paraffin. The kidney sections were dewaxed and rehydrated. After being blocked in hydrogen peroxide (3%) for 20 min, the sections were subjected to antigen retrieval. Then, the tissue sections were stained with hematoxylin and eosin (H&E) to evaluate histopathological changes. For immunohistochemical staining, the dewaxed sections were blocked in 3% hydrogen peroxide and incubated with anti-IRE1α (Novus Biologicals, Littleton, CO, USA), anti-LC3 (MBL, Nagoya, Japan), anti-ET-1 (ABclonal Inc., Boston, MA, USA), anti-ET-2 (Bioss antibodies Inc., Woburn, MA, USA) or anti-NLRP3 (Abcam, Cambridge, MA, USA) antibodies at room temperature for 2 h. Then, the slides were incubated with a secondary antibody at room temperature for 1 h, and a STARR TREK Universal HRP detection kit (Biocare Medical, Concord, CA, USA) was used. Finally, the slides were stained with hematoxylin and observed using a light microscope. The images were quantified the positive cells by ImageJ plugins. The IHC of positive percentage areas were analyzed in 10 fields of view.

The kidneys were fixed in 10% formalin at room temperature for 72 h, then dehydrated and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) for histological evaluation. The tubular injury rate of 20 contiguous fields per kidney (5 mice per group) was analyzed. The severity of tubular damage was graded from 0 to 5 according to tubular changes, such as tubular dilatation, flattening of the tubular epithelium and loss of brush borders. The tubular injury score was graded as follows: 0, normal; 1, lesion area <10%; 2, lesion area between 10 and 20%; 3, lesion area between 20 and 30%; 4, lesion area between 30 and 40%; and 5, lesions involving >40% of the field.
For immunohistochemical (IHC) staining, the slides were incubated for 2 h at room temperature with anti-LC3 (MBL, Nagoya, Japan), anti-α7nAChR (Proteintech Group, Chicago, IL, USA), anti-IRE1α (Novus Biologicals, Littleton, CO, USA), anti-KIM-1 (Novus Biologicals, Littleton, CO, USA) or anti-NLRP6 (Bioss Antibodies Inc., Woburn, MA, USA) antibodies. The slides were added with a secondary antibody for 1 h and were displayed using a STARR TREK Universal HRP detection kit (Biocare Medical, Concord, CA, USA). Finally, the slides were stained using hematoxylin.