Image analysis software

Cells were lysed with lysis buffer (10 mM Tris/100 mM NaCl/5 mM EDTA/10% Glycerol/1% triton x‐100/1% NP‐40/0.1% SDS/ Protein Inhibitor Cocktail 1:100) and then detected using a Bradford method to quantify its protein concentration. Consequently, 30 or 40 μg of lysate protein was run on 10% SDS‐PAGE gel and transferred to the PVDF membrane. The membranes were blocked in 5% non‐fat milk dissolved in 1 × TBS buffer at room temperature for 1 h and then incubated with the primary antibody at 4°C overnight. After being washed three times in TBST, the blots were incubated with horseradish peroxidase (HRP)‐coupled secondary antibodies at a dilution of 1:10000 for 1 h at room temperature. Western blot analysis was conducted by using ECL reagent (Pierce) and detected using image analysis software (Bio‐Rad Laboratories). The membranes were re‐probed with internal reference antibodies, such as ERK2, P38, Smad2/3 or Tubulin, after probed with antibodies against phospho‐ERK1/2, phospho‐JNK, phospho‐P38, phospho‐Smad2/Smad3 or αSMA, TAGLN and COL1A1. The expression of the protein was normalized to tubulin and expressed as fold change of control.

The expression of Bax, Bcl-2, cleaved caspase-3, PI3K, p38MAPK, eNOS and iNOS was analyzed by western blot analysis, and samples was normalized to GAPDH, as previously described [59 (link)–61 (link)]. The myocardium were lysed utilizing the RIPA buffer (1 ml/100 mg) at 0° C, and centrifugated at the speed of 12000 r/min for 10 min. The supernatant (40 μg) was loaded to western blot assay by using 15% SDS-PAGE, and was electronic-transferred onto the polyvinylidene fluoride (PVDF) membrane (Dupont, USA). Then, the membranes were blocked utilizing 5% non-fat milk for 2 h at room temperature, followed by wash with PBST buffer for 3 times (5 min/wash). The membranes were incubated with primary antibodies at 4° C for whole night. The membranes were washed with PBST buffer for three times (5 min /time). Then, the membranes were cultivated with AP-labelled goat anti-rabbit IgG and AP-labeled goat anti-mouse IgG (1:10000, Santa Cruz, Co. Ltd. CA, USA) at 37° C for 60 min, and rinsed with PBST buffer for three times (5 min /time). Finally, the membranes were incubated with ECL reagent (Pierce, USA) in the dark for 3 min. The density of bands was evaluated by Image Analysis Software (Bio-Rad Co. Ltd., USA).

MDCK cells exposed to specified concentrations of oxalate and with or without RSG and GW9662 were harvested and homogenized in lysis buffer containing proteinase inhibitors and phosphatase inhibitors (Beyotime Institute of Biotechnology, Shanghai, China). Total protein concentrations of the supernatants were determined with a BCA protein assay (Beyotime Institute of Biotechnology, Shanghai, China). A total of 25 μg of protein was separated by 8% or 10% SDS-PAGE and electrophoretically transferred onto 0.45 μm PVDF membranes. After blocking in 5% nonfat milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The membranes were then incubated with a horseradish peroxidase- (HRP-) conjugated secondary antibody for 1 h at room temperature. Finally, the protein bands were visualized with an enhanced chemiluminescence (ECL) Western blotting reagent. The blots were analyzed using Bio-Rad image analysis software, and the results are representative of at least three independent experiments. Proteins from samples from patients with urolithiasis and mice were extracted and homogenized with a homogenizer, and the subsequent steps were the same as above.

Frozen tumor specimens were treated in radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS) containing a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) for 30 min on ice. Equal amounts of protein were separated by 10% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich Shanghai Trading Co., Ltd., Shanghai, China). After blocking with 5% skim milk solution for 2 h, the membranes were incubated with a rabbit polyclonal plexin-B3 antibody (1:200; cat. no. sc-67144, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or a mouse monoclonal β-actin antibody (1:1,000; cat. no. 60008-1-Ig, Proteintech, Chicago, IL, USA) at 4°C overnight. The membranes were then incubated with appropriate secondary antibodies: Goat anti-rabbit (cat. no. sc-2004) or goat anti-mouse (cat. no. sc-2005) horseradish peroxidase (HRP)-conjugated immunoglobulin G (1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature, and the signal of the protein was revealed using an enhanced chemiluminescence method (Auragene Bioscience, Inc., Changsha, China). Band intensities were quantified using image analysis software (Bio-Rad Laboratories, Hercules, CA, USA). Each experiment was repeated a minimum of three times.