P16ink4a

Whole cell lysates were extracted by using the lysis buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton, and 10% glycerol along with protease and phosphatase inhibitor cocktail (4693132001& 4906837001, Roche, Basel, Switzerland) protein concentrations were determined by the Bradford assay. Soluble proteins (30–40 μg) were subjected to SDS–polyacrylamide gel electrophoresis. Separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and immunoblotted with anti-GATA4 (ab134057, 1:2000, Epitomics, Burlingame, CA, USA), -FLAG (F1804, 1:2000, Sigma), -CyclinD1 (2978S, 1:2000, CST, Danvers, MA, USA), -KRAS (12063-1-AP, 1:1000, Proteintech, Chicago, IL, USA), -Phos-ERK1/2 (4376S, 1:2000, CST), -Phos-AKT1 (3787S, 1:2000, CST), -ERK1/2 (4695S, 1:2000, CST), -AKT1 (2973S, 1:2000, CST), -PTEN (9188s, 1:1000 CST), -c-MYC (M4439, 1:2000, Sigma), -p21(2947S, 1:1000, CST), -p27(3686S, 1:1000, CST), -p53(2524S, 1:1000, CST), -p14/ARF(2407, 1:1000, CST), -p16/Ink4a (3562-1, 1:1000, Epitomics), -p15 (YT3492, 1:1000, ImmunoWay, Newark, DE, USA) or β-actin (A5316, 1:2000, Sigma) antibody. Immunoreactive proteins were visualized using ECL Western Blotting Substrate (PREGENE, Beijing, China) and X-ray films.

Formalin-fixed, paraffin-embedded kidney specimens were prepared as 3-μm-thick sections. For general histology, sections were processed for Masson trichrome staining according to the manufacturer’s instructions (Solarbio Life Science, Beijing, China). The collagen volume fractions of Masson staining were quantified by the ImageJ program (National Institutes of Health, Bethesda, MD). For immunohistochemistry staining, sections were incubated with primary antibody against TGFβ1 (Santa Cruz Biotechnology, TX, USA), p16^INK4A (Abcam, MA, USA), Serpin E1 (Santa Cruz Biotechnology), followed by incubation with secondary antibodies and the use of diaminobenzidine substrate kit (Servicebio, Shanghai, China). As negative controls, the primary antibodies were replaced by nonimmune serum from the same species and no nonspecific staining was observed. Computerized morphometry of immunohistochemical staining of TGFβ1, p16^INK4A and Serpin E1 was performed as described previously^{18 (link)} by using Image-Pro Plus software (Media Cybernetics).

Cells were lysed in radioimmunoprecipitation assay buffer supplemented with the protease inhibitor and phenylmethylsulfonyl fluoride. Cell lysates were subjected to western immunoblot analysis as described before^{20 (link)}. The blots were incubated with specific antibodies against E-cadherin (Cell Signaling Technology), fibronectin (Santa Cruz Biotechnology), Serpin E1(Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), p16^INK4A (Abcam), and GAPDH (Santa Cruz Biotechnology). The integrated pixel density of immunoblot bands was determined using the ImageJ program.

Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies, GGCT (6-1E, Cosmo Bio, Tokyo, Japan); p21^WAF1/CIP1 (BD biosciences, New Jersey, NJ, USA); Caspase-8 (Cell Signaling Technology, Danvers, MA, USA); β-actin and GAPDH (Wako Pure Chemical Industries, Osaka, Japan); rabbit monoclonal antibodies, p16^INK4A (Abcam, Cambridge, MA, USA); PARP-1 (Enzo Life Science, Farmingdale, NY, USA); Caspase-3 (Cell Signaling Technology); and p53 (Cell Signaling Technology). Horse anti-mouse IgG-horseradish peroxidase (HRP) conjugates were from Vector Laboratories (Burlingame, CA, USA). Goat anti-rabbit IgG–HRP was from Jackson Immuno Research Laboratories (West Grove, PA, USA).

Antibodies against p53, p21, p16^INK4a and β-actin were purchased from Abcam (Cambridge, UK). Antibodies against p-eNOS and eNOS were purchased from Cell Signaling Technology (Boston, MA, USA).

Cells pellets were lysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA pH 8, 0.5% v/v Nonidet P-40, 0.1% (w/v) SDS, 0.5% (v/v) Sarkosyl) supplemented with 5 µg/ml protease inhibitors cocktail and 1 mM PMSF. Total protein content was determined by Bradford protein assay, and equal amounts of protein were separated in 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore Co., Bedford, MA). Transference was confirmed staining the membrane with ATX Ponceau S red staining solution (Sigma-Aldrich), followed by blocking with 5% milk in TBS (Tris-Buffered-Saline; 20 mM Tris, 150 mM NaCl) and 0.05% Tween-20 (Sigma-Aldrich). Primary antibody incubation was performed O/N at 4 °C with rotation, and HRP-secondary probing at RT for 1 h. Pierce^TM ECL Western Blotting Substrate in an Amersham Imager 600 (GE Healthcare) was used. The following primary antibodies were used: α-tubulin (Sigma-Aldrich, T9026; 1:10,000), Cx43 (Sigma-Aldrich, C6219; 1:1000), Twist-1 (Santa Cruz Biotechnology, sc-81417; 1:100), p16^INK4a (Abcam, ab108349; 1:100), p53 (Santa Cruz Biotechnology, sc-126; 1:100), p21 (Santa Cruz Biotechnology, sc-6246; 1:100), ERK (Santa Cruz; 1:200) NF-κB (Santa Cruz Biotechnology, sc-8008; 1:100) and lamin A (Santa Cruz Biotechnology, sc-20680; 1:1000). A minimum of n = 3 experiment were performed.