Anti mouse secondary antibodies

The mouse kidney tissue was lysed with Protein Extraction Reagent (cat. no. 78505; Thermo Fisher Scientific, Inc.) containing protease-inhibitor (cat. no. P8340, Sigma-Aldrich; Merck KGaA) and the protein concentration was quantified using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.) and 30 µg protein from each group was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto a polyvinylidene fluoride membrane and blocked with 5% milk for 1 h. The membranes were then incubated with the following primary antibodies overnight at 4°C: Anti-VCAM-1 (0.25 µg/ml, cat. no. AF643; R&D systems), anti-HO-1 (1:1,000, cat. no. 27282-1-AP; ProteinTech Group, Inc.), anti-cleaved caspase-3 (1:1,000, cat. no. 9664; Cell Signaling Technology, Inc.) and anti-β-actin (1:1,000, cat. no. 58169; Cell Signaling Technology, Inc.), followed by the horseradish peroxidase-conjugated anti-goat (1:2,000, cat. no. ab97110; Abcam), anti-rabbit (1:2,000, cat. no. 7074; Cell Signaling Technology, Inc.) or anti-mouse secondary antibodies (1:2,000, cat. no. 7076; Cell Signaling Technology, Inc.) at room temperature for 1 h. Protein bands were visualized using an ECL reagent (Beyotime Institute of Biotechnology) and analyzed using densitometry with ImageJ software (version 1.48; National Institutes of Health).

NHDF cells were
treated with test samples and then lysed in RIPA buffer (Nacalai Tesque,
Inc., Japan, #16488-34) mixed with 1% phosphatase inhibitor cocktail
(Nacalai Tesque, Inc., Japan, #07575-51). Total cellular protein samples
were boiled in 5 × SDS sample buffer for 5 min, separated by
10% SDS-PAGE, and transferred to Immobilon-P PVDF membrane (Merck
Millipore, United States, #IPFL00010). The blot was incubated with
a blocking solution (2% BSA in TBS/T solution) and then probed with
anti-pCREB (1:1000, Cell Signaling Technology, United States, #9198S)
or anti-GAPDH (1:3000, Thermo Fisher Scientific, United States, #MA5-15738).
Horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signaling
Technology, United States, #7074) or anti-mouse secondary antibodies
(Cell Signaling Technology, United States, #7076) were used at a 1:3000
dilution. Immunoreactive bands were visualized using immobilon western
chemiluminescent HRP substrate (Merck Millipore, United States, #WBKLS0500)
using LuminoGraphI (WSE-6100, ATTO Corporation, Japan).

Human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell Research Laboratories, Inc. and cultured in EC medium containing EC growth supplement (ScienCell Research Laboratories, Inc.), 5% fetal bovine serum (FBS; ScienCell Research Laboratories, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin in 37°C. HUVECs at passages 3–5 were used for all experiments. Tan 2A was purchased from Selleck Chemicals (Cat. no. S2365, Lot no. S2767130005001). DMSO (sigma-Aldrich; Merck KGaA) was used as vehicle control. Lipofectamine™ RNAiMAX Transfection Reagent and Lipofectamine^® 3000 Transfection Reagent were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Antibody against GLUT-1 (ab115730, 1:2,000) was purchased from Abcam. Antibodies against HIF-1α (#36169, 1:1,000), RBPJκ (#5313, 1:1,000) and GAPDH (#5174, 1:2,000) and horseradish peroxidase-conjugated goat anti-rabbit (#7074, 1:2,000) or anti-mouse secondary antibodies (#7076, 1:2,000) were purchased from Cell Signaling Technology, Inc.