Total proteins were extracted from frozen LV tissue and cultured PVFs using RIPA lysis buffer. Proteins were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). All sample proteins loaded equally (20 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Subsequently, the PVDF membrane was blocked with TBST containing 5% BSA for 1 h and incubated at 4°C overnight with a primary antibody. Then, the membrane was washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. The primary antibodies used were as follows: GAPDH (1 : 5000, 60004-1-Ig, Proteintech), CTGF (1 : 1000, sc-365970, Santa Cruz), α-SMA (1 : 1000, ab7817, Abcam), Col 1 (1 : 1000, ab34710, Abcam), Col 3 (1 : 1000, ab7778, Abcam), TGF-β1 (1 : 1000, ab215715, Abcam), TGFR-2 (1 : 1000, ab269279, Abcam), Smad2 (1 : 1000, ab33875, Abcam), Smad3 (1 : 1000, ab40854, Abcam), and phospho-Smad2/3 (1 : 1000, ab272332, Abcam). Immunoblots were quantified using a Tanon 5200 Multi Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd., Shanghai, China).
Ab33875
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab33875 is a laboratory equipment product manufactured by Abcam. It is designed for general laboratory use. The core function of this product is to provide a tool for researchers and scientists in their work. No further details about its intended use or application are available.
Automatically generated - may contain errors
Lab products found in correlation
Ab40854, by Abcam (6 mentions)
Ab52903, by Abcam (5 mentions)
Ab188334, by Abcam (4 mentions)
Ab53100, by Abcam (3 mentions)
Ab5103, by Abcam (3 mentions)
Ab34710, by Abcam (2 mentions)
Ab215715, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab17942, by Abcam (2 mentions)
Ab179695, by Abcam (2 mentions)
Ab16502, by Abcam (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ab97779, by Abcam (2 mentions)
Ab181603, by Abcam (2 mentions)
Ab40759, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab280888, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab197896, by Abcam (2 mentions)
Ab28364, by Abcam (2 mentions)
18 protocols using ab33875
1
Western Blot Analysis of Cardiac Fibrosis Markers
2
Immunoblotting for Protein Expression Analysis
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Vascular tissues and cells were prepared according to immunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology) instructions. Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo, USA). We used 30–40 μg protein for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred the proteins to nitrocellulose membranes (GE Healthcare, USA)8 (link). We then blocked the membranes with 5% skin milk in phosphate-buffered saline (PBS) and probed them with the following primary antibodies overnight at 4 °C: ERK1/2 (ab17942, Abcam), pERK1/2 (9102S, CST), TGF-β (ab179695, Abcam), Smad2 (ab33875, Abcam), pSmad2 (ab188334, Abcam), NF-κB p65 (ab16502, Abcam), IκBα (4812S, CST), pIκBα (2859S, CST), β actin (4970S, CST), and Lamin b1 (13435S, CST), followed by horseradish peroxidase-conjugated secondary antibodies to rabbit IgG for 1 h at 37 °C. Blots were visualized using an enhanced chemiluminescence kit (ECL; Thermo Scientific, USA)8 (link). All original blots are shown in the supplementary material, but the original blots in Fig. 4 d for Lamin b1, Fig. 6 for Smad 2, S2A and S4B were not saved, and only the images cropped after scanning were saved.
3
Western Blot Analysis of EMT Markers
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Treated cells were lysed in RIPA buffer supplemented with phenylmethylsulphonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China). The BCA method was used to assess protein concentration. Proteins were separated by 12.5% SDS-PAGE and transferred to PVDF membrane and immunoblotted with the following antibodies: anti-SMAD2 (1:1000, ab33875, Abcam, Cambridge, MA, USA), anti-Snail (1:1000, Abcam, ab53519), anti-E-cadherin 1:1000, (1:1000, Abcam, ab231303), anti-MMP-2 (1:1000, Abcam, ab97779), anti-MMP9 (1:1000, Abcam, ab137867), anti-TIMP2 (Abcam, ab180630), and anti-GAPDH (1:1000, Abcam, ab181603). After rinsed with TBST, the membranes were incubated with HRP-conjugated secondary antibody (1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology), developed with an ehanced chemiluminescence regent (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and visualized by Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA). The experiments were performed in triplicate.
4
Comprehensive Protein Expression Analysis
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Total protein was extracted followed by concentration measurement utilizing a bicinchoninic acid kit. Protein (50 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane which was then blocked with 5% skimmed milk powder at ambient temperature for 1 h. Next, the membrane was probed with diluted primary rabbit antibodies to Smad2 (ab33875, 1: 1000, Abcam), Smad4 (ab40759, 1: 5000, Abcam), HDAC3 (ab7030, 1: 500, Abcam), TGIF1 (ab52955, 1: 5000, Abcam), cleaved caspase-3 (ab32042, 1: 1000, Abcam), total caspase-3 (ab32150, 1: 1000, Abcam), MMP-2 (ab97779, 1: 2000, Abcam), MMP-9 (ab38898, 1: 1000, Abcam), TGF-βRII (sc-17791, 1: 1000, Santa Cruz Biotechnology, Santa Cruz, CA) and GAPDH (ab9485, 1: 2500, Abcam) overnight at 4ºC. The next day, the membrane was re-probed with HRP-labeled secondary antibody of goat anti-rabbit antibody to IgG H&L (ab97051, 1: 2000, Abcam) for 1 h. The results were visualized utilizing enhanced chemiluminescence reagents (BB-3501, Ameshame, Little Chalfont, UK). Images were acquired through Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA), followed by analysis using Quantity One v4.6.2 software. The relative protein level was described as the ratio of gray value of protein to be tested to that of GAPDH [22 , 23 (link)].
5
Western Blot Analysis of Cell Signaling
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The cells, after treatment, were collected and lysed in RIPA lysis buffer. Then, the cell lysates of different groups were collected. Total protein was separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Then the membranes, after blocking with 5% dry nonfat milk, were incubated with the following primary antibodies: VCAM-1 (#ab174279, Abcam), sE-selectin (Anyan, Shanghai), bcl2 (#ab182858,Abcam), p53 (#ab32389, Abcam), p21 (#ab109520, Abcam), p16 (#ab51243, Abcam), TGFβRI (#ab121024, Abcam), TGFβRII (#ab186838, Abcam), Smad2 (#ab33875, Abcam), Smad3 (#ab40854, Abcam), pSmad2 (ab216482, Abcam), and pSmad3 (#ab52903, Abcam). Thereafter, the membranes were incubated with the secondary antibody (goat anti-rabbit IgG H&L (Alexa Fluor® 488), #ab150077). Image Lab™ Software (Bio-Rad, Shanghai, China) was applied for quantifying the intensity of the bands.
6
Protein Expression Analysis in Heart Tissue
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Fresh heart tissue was added to RIPA lysis buffer to obtain total protein. Protein (25 μg) was subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with skim milk for 30 min at room temperature and then incubated with primary antibodies at 4°C overnight. The next day, after washing with TBST, the membranes were incubated with conjugated secondary antibodies (1:3000, CST) at room temperature for 1 h, and the bands were visualized by chemiluminescence. The main antibodies used in this study were: anti-TGF-β (ab215715, Abcam), anti-p-Smad2 (ab280888, Abcam), anti-Smad2 (ab33875, Abcam), anti-p-AKT (ab38449, Abcam), anti-AKT (orb11276, Biorbyt), anti-p-ERK1/2 (ab214036, Abcam), anti-ERK1/2 (ab184699, Abcam), anti-p-IKKα (ab138426, Abcam), anti-IKKα (ab32041, Abcam), anti-p-P65 (ab76302, Abcam), anti-P65 (ab16502, Abcam), and anti-GAPDH (ab8245, Abcam).
7
Protein Isolation and Western Blot Analysis
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Total protein was isolated from OS cells and xenograft tumor tissues by Radio Immunoprecipitation Assay lysis buffer. After separating by 10% sodium dodecyl sulfate polyacrylamide gel, proteins were then moved to polyvinylidene fluoride membranes (Millipore, United States). Then, the membranes were blocked with 5% nonfat milk, incubated with primary antibodies against GAPDH (ab181602, 1:10,000; Abcam, Shanghai, China), transforming growth factor-β1 (TGF-β1; ab179695, 1:1,000), SMAD2 (ab33875, 1:1,000), SMAD3 (ab40854, 1:1,000), Smad2 (phospho S255) (ab188334, 1:2,000), Smad2 (phospho S467) (ab53100, 1:500), Smad3 (phospho T179) (ab193297, 1:1,000), Smad3 (phospho S213) (ab63403, 1:1,000), Smad3 (phospho S423/S425) (ab52903, 1:2,000), and Ki67 (ab16667, 1:1,000) at 4°C overnight, followed by incubation with secondary antibody for 1 h at darkness. The relative levels of proteins were evaluated using an ECL chemiluminescent detection system (Thermo Fisher Scientific, Rochester. NY).
8
Western Blot Analysis of Smad Proteins
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The BCA kit was used for determining the protein concentration. Next, proteins were then loaded on 10% SDS-PAGE and blotted onto a PVDF membrane. Next, the membrane was probed overnight at 4°C with the primary antibodies: anti-Smad2 (no. ab33875, 1:1000; Abcam), anti-p-Smad2 (no. ab188334, 1:1000; Abcam), anti-Smad3 (no. 66516-1-Ig, 1:1000; Proteintech), anti-p- Smad3 (no, ab52903, 1:1000; Abcam), and anti-β-actin (no. 66009-1-I,1:1000; Proteintech), followed by incubation with a secondary antibody (no. A0208, 1:2000; Beyotime Institute of Technology) for 50 min at room temperature. Thereafter, the blots were developed with an ECL kit. This assay was repeated in triplicate.
9
Quantifying SHCBP1 Expression in Cell Lines
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All the antibodies used in this study are shown as follows:
| Antibody | Information |
|---|---|
| SHCBP1 | 1:200 for IHC; 1:2000 for Immunoblot, SAB1307183, Sigma-Aldrich, MO |
| β-actin | 1:2500, ab8227; Abcam, UK |
| p-Smad2 | 1:2500, ab53100; Abcam, UK |
| Smad2 | 1:2000, ab33875; Abcam, UK |
| p-Smad3 | 1:2500, ab52903; Abcam, UK |
| Smad3 | 1:2500, ab40854; Abcam, UK |
| p-ERK1/2 | 1:2500, ab214362; Abcam, UK |
| ERK1/2 | 1:1500, ab17942, Abcam, UK |
| Primers | Sequences |
|---|---|
| SHCBP1-FP | 5′-CCCTTCTGAGCAAGTCGAGG-3′ |
| SHCBP1-RP | 5′-AACTGGTTCCCCACAATCCC-3′ |
| GAPDH-FP | 5′-CGACCACTTCGTCAAGCTCA-3′ |
| GAPDH-RP | 5′-GGTGGAGCACAGGGTACCTTATT-3′ |
10
Comprehensive Stem Cell Marker Analysis
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The following primary antibodies were used in the study including anti-CXCR4 (ab124824, AbCam), anti-TRAIL (AF1121, R&D), anti-c-Kit (AF1356, Novus Biological), anti-CD47 (ab175388, Abcam), anti-Fasl (ab15285, Abcam), anti-PDL1 (NBP1-76769, Novus Biological), anti-CD4 (ab183685, Abcam), anti-SSEA1 (ab16285, Abcam), anti-Oct4 (ab184665, Abcam), anti-Sox2 (MAB2018R-100, R&D), anti-Nanos3 (ab70001, Abcam), anti-Ifitm3 (ab109429, Abcam), anti-Stellar (Invitrogen, PA5-34601), anti-PRDM14 (ab187881, Abcam), anti-Vasa (ab27591, Abcam), anti-DAZL (NB100-2437, Novus biologicals), anti-SMAD2 (ab33875, Abcam).
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