Anti twist1

The protein expression levels of OPN, Vimentin, E-cadherin, N-cadherin, Twist1, AXL and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were evaluated by Western blot. Total protein was extracted from cells by lysing the cells in RIPA buffer [50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% NP-40, 0.25% Na-deoxydiolate, 1 mM EDTA] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 mM Na₃VO₄, 1 Mm NaF). Protein samples was separated by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred onto PVDF (polyvinylidene fluoride) membranes. After blocked with 5% non-fat milk/TBST, the membrane was incubated with the primary antibody. The following primary antibodies were used: anti-OPN (Abcam, Cambridge, UK), anti-Vimentin (Abcam), anti E-cadherin (Cell Signal Tech, Danvers, USA), anti-N-cadherin (Millipore, Massachusetts, USA), anti-Twist1 (Abcam), anti-Snail (Cell Signal Tech), anti-AXL (Abcam), or anti-GAPDH (Cell Signal Tech); and detected with enhanced chemiluminescence reagents (Thermo Fisher Scientific). Bands were acquired by Molecular Imager ChemiDox XRS+ Imaging System with Quantity One Image software (Bio-Rad Laboratories).

Serial sections (4 μm) subjected to immunohistological staining were fixed with freshly prepared 3 % H₂O₂ with 0.1 % sodium azide to quench endogenous peroxidase and then treated with antigen retrieval solution for 15 min. After placing in blocking reagent for 15 min, the sections were incubated in primary anti-HFI-2α (1:500, Abcam), anti-E-cadherin (1:1000, Abcam), anti-Twist1 (1:500, Abcam) and anti-Twist2 (1:350, Abcam) monoclonal antibody overnight at 4 °C, followed by incubation with the secondary antibody and Extravidin-conjugated horseradish peroxidase. The staining intensity was scored as: 0 (<10 %), 1 (10–50 %), 2 (>50 %). The final score was calculated by multiplication of the intensity score the quantity score. A score ≥ 2 was considered to represent positive expression.

TGF-β was purchased from PeproTech (USA). The antibodies used were as follows: anti-Twist1 was purchased from Abcam (Cambridge, UK); anti-SNCG and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Smad2 and anti-Smad3 were purchased from ABclonal (Wuhan, China); anti-phospho-Smad2 (S245/250/255) and anti-phospho-Smad3 (S423/425) were from Cell Signaling Technology (Danvers, MA, USA). All siRNAs were custom-synthesized products of Ribobio Co., Ltd. (Guangzhou, China). The siR-Ribo negative control (siControl) was used for all siRNA experiments. The target sequences for Twist1 knockdown are as follows: 5′-GGUACAUCGACUUCCUCUA-3′ for siTwist1#1, and 5′-UUGAGGGUCUGAAUCUUGCUCAGCU-3′ for siTwist1#2. The sequence of siRNA targeting the 3′-untranslated region of Twist1 gene (siTwist1#3) is 5′-CACCTCTGCATTCTGATAGAA-3′. The two target sequences for SNCG knockdown are as follows: 5′-GCAGCTGAGAAGACCAAGG-3′ for siSNCG#1 and 5′-GGAGAATGTTGTACAGAGC-3′ for siSNCG#2. Target sequences for Smad3 are 5′-GGAGAAAUGGUGCGAGAAG-3′.

Cells were collected and lysed in lysis buffer on ice. Total proteins were separated by 10 % SDS-PAGE and blotted on PVDF membrane. Membranes were blocked with 10 % non-fat milk powder at room temperature for 2 h and incubated with primary antibodies: anti-HIF2α (1:200), anti-VE-cadherin (1:1000), anti-MMP2 (1:1000), anti-MMP9 (1:5000), anti-Twist1 (1:200), anti-Twist2 (1:50) (all from Abcam, Cambridge, UK) and anti-GAPDH (1:1000, Santa Cruz Biotechnology, CA, USA), at 4̊C overnight. After three washes, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000; Santa Cruz Biotechnology). Reactive bands were detected using ECL western blotting detection reagent (GE Healthcare, USA).

Western blot analyses were performed as previously described [7 (link)]. List of antibodies used: anti-LY75 (Abcam, Branford, CT, USA and Santa Cruz Biotechnology Dallas, TX, USA), anti-Snail1, anti-FN1, anti-E-cad, anti-EpCAM, anti-AXIN1, anti-WNT3, anti-EVX2, anti-IFFO1, anti-CLDN5, anti-HOOK1, anti-JMJD8, anti-RAMP2, anti-KLF-4, anti-β-actin antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and anti-Twist1, anti-E-cadherin, anti-N-cadherin, anti-APC2, and anti-Cend1 antibodies (Abcam Branford, CT, USA).

Proteins were extracted using 1% SDS lysis buffer. The concentration of proteins was measured with the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Each sample was separated by SDS-PAGE gel, transferred into nitrocellulose membrane (Millipore Corporation, Billerica, MA, USA). Membranes were incubated with the following primary antibodies: anti-PPP3CB (Abcam, Cambridge, UK), anti-E-cadherin (Bioworld, Nanjing, China), anti-Vimentin (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (Abcam, Cambridge, UK), anti-Twist1(Abcam, Cambridge, UK),anti-Snail1(Cell Signaling Technology, Danvers, MA, USA),anti-GFP (Bioworld, Nanjing, China), anti-β-actin (Transgene, Beijing, China). The target bands were detected by ECL (Millipore Corporation, Billerica, MA, USA).

Protein expression was detected by Western blot analysis as described previously (35 (link)). Briefly, the isolated glomeruli were homogenized in RIPA buffer (MilliporeSigma). The supernatants were collated after centrifugation at 13,000g at 4°C for 30 minutes. Concentrations of protein were quantitated using the DC Protein Assay Kit (Bio-Rad). Equal amounts of sample were subjected to electrophoresis through 4%–12% Bis-Tris gels and transferred to polyvinylidene difluoride membranes. After blocking with 5% milk in Tris-buffered saline/Tween 20, the blots were incubated with anti-Twist1 (Abcam, catalog 50887), anti-nephrin (Thermo Fisher Scientific, catalog PA5-91907), anti-podocin (MilliporeSigma, catalog P0372), or anti-GAPDH (Cell Signaling Technology [CST], catalog 2118) overnight at 4°C. The blots were then washed and incubated for 1 hour at room temperature with respective secondary antibodies accordingly (CST, catalog 7074 and 7076, or Abcam, catalog ab6741). Bands were detected using an enhanced chemiluminescence detection system (BiO-RAD ChemiDoc MP Imaging System). The detected bands were quantified by densitometry through ImageJ 1.38 for Windows (NIH).