For protein extract preparation, cells were lysed on ice with RIPA Lysis Buffer (ThermoFisher) containing complete protease and phosphatase inhibitor cocktail (Roche). Soluble protein extracts were separated by centrifugation at 13000 rpm for 15 min and diluted in Laemlli sample buffer. The obtained cell lysates were resolved on sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and transferred on polyvinylidene difluoride membrane Hybond TM-P (Amersham Bioscience). Membranes were saturated with 5% bovine serum albumin at room temperature for 2 h and incubated with the following primary antibodies at 4 °C overnight. Primary antibodies were used as follows: ELF3 (1:1000, AF5787; R&D Systems, Minneapolis, USA), EHF (1:1000, 27195-1-AP; Proteintech, Illinois, USA), TGIF1 (1:1000, ab52955; Abcam, Masseachusettes, USA), β-actin (1:1000, 3700S; CST, Boston, USA) and GAPDH (1:1000, 2118S; CST, Boston, USA). Secondary anti-mouse IgG (ab175775), anti-rabbit IgG (ab175773), and anti-goat IgG (ab175776) all conjugated to Alexa Fluor 680 (Abcam, Masseachusettes, USA) were incubated with the membranes for 2 h at room temperature at 1:10,000 dilution. All bands of western blot were detected and qualified with gray scale ratio by Odyssey CLx imaging systems (LI-COR, Nebraska, USA).
Ab52955
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab52955 is a laboratory equipment product offered by Abcam. It is a device used for a specific laboratory function. No further details about its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 680, by Abcam (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Complete protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Hybond p, by Cytiva (1 mentions)
Ab175776, by Abcam (1 mentions)
Ab175775, by Abcam (1 mentions)
Ab175773, by Abcam (1 mentions)
Hybond, by Cytiva (1 mentions)
Ab32042, by Abcam (1 mentions)
Image analysis system, by Bio-Rad (1 mentions)
Ab97779, by Abcam (1 mentions)
Ab7030, by Abcam (1 mentions)
Ab32150, by Abcam (1 mentions)
Ab33875, by Abcam (1 mentions)
Ab40759, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab38898, by Abcam (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
4 protocols using ab52955
1
Protein Extraction and Western Blotting
2
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)].
3
Immunocytochemical Staining of CtBP2 and TGIF
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Staining reagents included CtBP2 (sc-17759; 1:200; Santa Cruz Biotechnology, Inc.) and TGIF (ab52955; 1:1,000; Abcam) primary antibodies, anti-mouse (SAB3701092) or anti-rabbit (F4890) secondary antibodies (MilliporeSigma), and Hoechst 33342 (Beyotime Institute of Biotechnology). The cells were digested with trypsin and pipetted vigorously to make a single-cell suspension. The cells were placed on glass coverslips (24-well plates), incubated at 37°C and fixed with 4% PFA for 20 min at 15–25°C. The glass coverslips were then washed three times with PBS for 10 min each and blocked with 10% bovine serum albumin (BSA) for 2 h at 15–25°C. Subsequently, all cells were incubated with primary antibody (1:200) overnight at 4°C. The following day, the cells were incubated with secondary antibody (1:2,000) for 2 h at 15–25°C. Finally, the cells were stained with Hoechst 33258 (Beyotime Institute of Biotechnology) at room temperature for 10 min, fixed with anti-fade solution and imaged under a fluorescence microscope (Zeiss AG).
4
Protein Isolation and Western Blot Analysis
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Proteins from tissues and cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland) and were harvested by centrifugation with 12,000 rpm for 10 min at 4°C. BCA Protein Assay Kit (Tiangen, Beijing, China) was used to quantify the protein concentrations. Equal amounts of proteins (15 mg) were separated through 10% SDS-PAGE gel and then transferred onto PVDF membranes. Next, the membranes were incubated with anti-TGIF1 (1:1000, ab52955, Abcam, Cambridge, MA) at 4°C overnight after blocked with 5% non-fat milk for 1 h at normal temperature and then subsequently incubated with HRP conjugated secondary antibody at room temperature for 2 h. Immunoreactive bands were developed using Enhanced chemiluminescence kit (Amersham, Little Chalfont, UK).
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