Multi gauge software of science lab 2006

Total proteins were extracted from heart tissues or endothelial cells using protein extraction reagents. Protein concentrations were quantified, and equal amounts of proteins (60 μg) were separated by 12% SDS-PAGE gel and electro-transferred to nitrocellulose membranes. After blocking with 5% skimmed milk for 2 h, membranes were incubated with the following primary antibodies at 4 °C overnight: NLRP3 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 19771-1-AP), caspase-1 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 22915-1-AP), GSDMD (Santa Cruz, USA, 1:500, Cat. No.: A2315), IL-1β (ABclonal, Boston, USA, 1:1000, Cat. No.: A1112), IL-18 (ABclonal, Boston, USA, 1:1000, Cat. No.: A1115), Transferrin (BIOSS, Beijing, China, 1:1000, Cat. No: bs-2052R), or GAPDH (Proteintech, Chicago, USA, 1:2000, Cat. No: 60004-1-lg). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibody (1:10,000) for 2 h. Western blot bands were analyzed with the ChemiDicTM XRS+ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and the band densities were quantified with Multi Gauge Software of Science Lab 2006 (FUJIFILM Corporation, Tokyo, Japan).

Total proteins were extracted from heart tissues or RAW264.7 cell lysates and supplemented with a cocktail of protease inhibitors and phosphatase inhibitors. Proteins were quantified to an equivalent amount, separated by SDS-PAGE gel, and electro-transferred to a PVDF membrane. Having been blocked in 5% skimmed milk, membranes were incubated with antibodies: anti-LFA-1 (ITGAL, Affinity Biosciences. DF5625, 1:1000), anti-p-JNK (Affinity Biosciences. AF3318, 1:1000), anti-total JNK (Affinity Biosciences. AF6138, 1:1000), anti-p-ERK (Affinity Biosciences. AF1015, 1:1000), anti-total ERK (Affinity Biosciences. AF0155, 1:1000), anti-p-p38(Affinity Biosciences. AF4001, 1:1000), anti-total p38 (Affinity Biosciences. AF6456, 1:1000) and GAPDH. The loading control was GAPDH (Proteintech, 60004-1-lg, 1:10000). Western blot bands were analyzed with the ChemiDicTM XRS + Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and the densities were quantified with Multi Gauge Software of Science Lab 2006 (FUJIFILM Corporation, Tokyo, Japan).

To assess the regulatory role of FGF10 on ER stress and apoptosis, the expression of relevant proteins was analyzed by western blot. For protein analysis of in vivo samples, total kidney tissues (contain both of cortex and medulla, but don't contain the renal fibrous capsule) were homogenized and total proteins were extracted using tissue lysis buffer. For protein analysis of in vitro samples, NRK-52E cultured in a petri dish was rinsed with PBS buffer three times; total proteins were extracted using cell lysis buffer. An equivalent of 100 μg protein of the in vivo sample (30 μg protein of the in vitro sample) was separated by Sodium Dodecyl Sulfate PolyAcrylamide and then transferred to a polyvinylidene fluoride (PVDF) membrane for immunoblot analysis. Primary antibodies against cleaved Caspase-3 (1:1,000), cleaved Caspase-9 (1:1,000), Bax (1:3,000), Bcl-2 (1:1,000), GRP78 (1:1,000), CHOP (1:5,000), XBP-1 (1:1,000), ATF-4 (1:1,000), ATF-6 (1:2,000), PDI (1:2,000), ERK1/2 (1:1,000), and phosphor-ERK1/2 (1:1,000) were used in the present study. GAPDH (1:2,500) was used as loading control. The signals were visualized with the ChemiDic™ XRS + Imaging System (Bio-Rad Laboratories). The band densities were quantified with Multi Gauge Software of Science Lab 2006 (FUJIFILM Corporation, Tokyo, Japan).