Las4000

The frozen muscle tissues (50 mg) were lysed using 300 µL of RIPA buffer (EzRIPA, ATTO, Tokyo, Japan) with proteinase and phosphatase inhibitors. The homogenized muscle tissues were sonicated and then centrifuged at 14,000× g for 15 min at 4 °C. The supernatants were carried on cleaned tubes. The total protein concentration of isolated protein was determined by a bicinchoninic acid assay kit (BCA kit; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The isolated proteins samples were separated on 8 or 10 percent sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane using a power station (WSE-3500, ATTO, Tokyo, Japan). Then, the membranes were blocked with 5% skim milk (SKI500, LPS solution, Daejeon, Korea) in tris buffered saline containing 0.1% tween-20 (TTBS) for 1 h at room temperature. After washing with TTBS, the membranes were incubated with diluted primary antibodies (Table S1). The probed membranes were rinsed with TTBS and loaded with appropriate peroxidase secondary antibodies. After a final wash with TTBS, the membranes were exposed with an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL, USA) by LAS-4000s (GE Healthcare, Chicago, IL, USA).

Fifty milligrams of frozen liver tissues was ground using mortar with liquid nitrogen and then incubated for 15 min on an ice container with added RIPA lysis buffer (EzRIPA; ATTO, Tokyo, Japan), mixed proteinase, and phosphatase inhibitors. After they were centrifuged at 14,000× g for 15 min at 4 °C, the supernatant was collected in a new tube, and the protein concentration was analyzed using a bicinchoninic acid assay kit (BCA kit; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then the proteins were transferred to polyvinylidene fluoride membranes using a power station (WSE-3500, ATTO, Tokyo, Japan). The membranes were incubated with diluted primary antibodies (Table S2) at 4 °C overnight. After washing three times with tris buffered saline (TBS) containing 0.1% Tween 20, the membranes were incubated with secondary antibodies for 2 h at room temperature. Finally, the membranes were developed by enhanced chemiluminescence using LAS-4000s (GE Healthcare, Chicago, IL, USA).

Eighty milligrams of frozen liver tissues were homogenized in 500 µL of RIPA buffer (EzRIPA, ATTO, Japan) containing proteinase and phosphatase inhibitors. The homogenized liver tissues were sonicated and centrifuged at 14,000× g for 15 min at 4 °C. The supernatants were transferred to cleaned tubes. The isolated proteins were quantified using a bicinchoninic acid assay kit (BCA kit; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Equal amounts of proteins were separated by 10 or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred to a polyvinylidene fluoride membrane using a power station (WSE-3500, ATTO, Osaka, Japan). Then, the membranes were incubated with 5% skim milk (SKI500, LPS solution, Daejeon, Korea) in Tris-buffered saline containing 0.1% Tween-20 (TTBS). After washing with TTBS, the membranes were incubated with diluted primary antibodies (as listed in Table S2). After washing three times with TTBS, the membranes were incubated with secondary antibodies. Then, the membranes were developed by chemiluminescence using LAS-4000s (GE Healthcare, Chicago, IL, USA).

To isolate protein from angiotensin II-treated TCMK-1 cells, the cells were scraped using the RIPA lysis buffer with phosphatase and proteinase inhibitor (EzRIPA; ATTO; Tokyo, Japan) and incubated on ice for 20 min. After centrifuging at 13,000× g for 20 min, at 4 °C, clean supernatants were moved to a new tube and the concentration of supernatants was analyzed with a bicinchoninic acid assay kit (BCA kit; Thermo Fisher Scientific, Inc.; Waltham, MA, USA). To validate protein expression from TCMK-1 cells, blotting was conducted. An equal amount of lysate proteins (30 μg/lane) was separated by 10% sodium dodecyl sulfate polyacrylamide gel using electrophoresis. Then, running proteins were transferred to polyvinylidene fluoride membranes, which were incubated with diluted primary antibodies (Anti-β-actin, AGTR1, TGF-β, SMAD2/3 and pSMAD2/3) at 4 °C. The incubated membranes with antibodies were thoroughly washed using Tris-buffered saline with 0.1% Tween 20 and incubated with secondary antibodies for 2 h at room temperature. All membranes were developed by enhanced chemiluminescence (LAS-4000s; GE Healthcare, Chicago, IL, USA). The antibodies information used in this study can be found in Table S2.

Total proteins were isolated from the whole frozen aorta using lysis buffer as previously described [28 (link)]. Afterwards, proteins were quantified using bicinchoninic acid assay (BCA) method (Thermo Fisher Scientific). A total 50 ug of proteins were loaded and separated on 10% polyacrylamide-SDS gels under reducing conditions. At the end of electrophoresis, proteins were transferred to nitrocellulose membranes (Amersham Bioscience, Buckinghamshire, UK). Membranes were blocked in TBS containing 0.1% Tween20 (TBST) and 5% dry non-fat milk (1 h at room temperature) and incubated with the different primary antibodies overnight at 4 °C. Next day, membranes were washed 10 min three times with TBST and incubated 1 h with the appropriate HRP (horseradish peroxidase)-conjugated secondary antibody (anti-rabbit, GENA934, anti-mouse, GENA931, Sigma Chemical [1/2000]) at room temperature. ECL kit (Amersham Bioscience) was used to develop. Results were analyzed by LAS 4000 (GE Healthcare Systems, Chicago, IL, USA) and the quantification of the bands density was done by using the Quantity One software (Bio-Rad, CA, USA). The following primary antibodies were employed p-SMAD2 (#3108, Cell signaling (1/1000)), p-SMAD3 (ab52903; abcam; (1/1000)), TβRII ((sc17792; Santa Cruz Biotechnologies; 1/300)) and TβRI (sc518086; Santa Cruz Biotechnologies; [1/300]).

We quantified the cytokine levels in the pooled rat serum, cell culture supernatants, and tumor extracts using the Proteome Profiler Rat Adipokine Array Kit (R&D Systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. To perform this analysis, we pooled an equal volume of serum from each rat. We then homogenized rat tumors in phosphate-buffered saline with a protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan) and 0.05% Triton-X (Sigma-Aldrich Japan, Tokyo, Japan). The homogenates were centrifuged at 20,000× g for 15 min at 4 °C, and the supernatants were used as tumor extracts. We mixed the pooled rat serum, cell culture supernatants, and tumor extracts with a cocktail of biotinylated detection antibodies. This mixture was incubated with a nitrocellulose membrane spotted with capture antibodies. Subsequently, we added streptavidin–horseradish peroxidase and chemiluminescent detection reagents to the membrane. The chemiluminescent signals from spots on the membrane were captured (LAS 4000; GE Healthcare Japan Corporation, Tokyo, Japan) and quantified using Image Gauge version 4.0 (GE Healthcare Japan Corporation, Tokyo, Japan).

The whole-cell lysates or the nuclear lysates were run on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and were transferred to polyvinylidene difluoride membrane using wet transfer. After the transfer, the membrane was incubated with 5% (w/v) bovine serum albumin (BSA) in TBST [20 mM Tris buffer pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20] for blocking. Then, it was incubated with various antibodies purchased from different companies. The antibodies against IκB, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), p65, c-Rel, and Lamin B1 were purchased from Santa Cruz Biotechnologies, USA. The antibodies against p-p38, p38, phosphorylated c-Jun N-terminal kinase (p-JNK), JNK, IκB kinase α/β (IKKα/β), Cullin-1, Cand-1, β-TrCP, Skp-1 and Rbx-1 were purchased from Cell Signaling Technologies, USA. The antibodies against His-tag, HA-tag, and proliferating cell nuclear antigen (PCNA) were purchased from Biolegend, USA. The antibody against Nedd8-Cullin was purchased from Abcam, UK. The antibody against LAMP-1 was purchased from Thermo Fisher Scientific, USA.
The horseradish-peroxidase-tagged secondary antibodies and antibody against glutathione-S-transferase (GST) were purchased from Sigma-Aldrich, USA. The immunoblots were developed using Clarity^TM ECL Substrate (Bio-Rad, USA) and detected using LAS 4000 (GE Healthcare Technologies, USA).