Cellular proteins were extracted using radio immunoprecipitation assay (RIPA) lysis buffer, and protease inhibitors were added to the lysate to protect the proteins. Anti-FTH polyclonal antibody (ab65080, Abcam), anti-αS1002/S100A2 (ab109494, Abcam), S100A4 (ab124805, Abcam), and S100P (ab133554, Abcam) monoclonal antibodies were diluted as the first antibody in a 1:1,000 ratio. Anti-mouse IgG H&L horseradish peroxidase (HRP), goat anti-rabbit IgG H&L HRP, and mouse anti-β actin monoclonal antibodies (Beijing Dingguo Chang Sheng Company) were diluted to a 1:2,500 ratio. Primary antibodies and polyvinylidene fluoride (PVDF) membranes were incubated overnight at 4 °C. The next day, the PVDF membranes were washed 3 times in phosphate-buffered saline (PBS) buffer. Then, they were incubated with secondary antibodies, which were labeled with HRP for 1 h. Target protein bands were determined by enhanced chemiluminescence, and β-actin was an internal reference to achieve consistent sample loading per well. Each experiment was repeated 3 times.
Ab133554
Manufactured by Abcam
Sourced in United Kingdom
Ab133554 is a rabbit monoclonal antibody that can be used to detect the target antigen. The antibody is purified and supplied in PBS buffer.
Automatically generated - may contain errors
Lab products found in correlation
Ab109494, by Abcam (1 mentions)
Ab65080, by Abcam (1 mentions)
Ab124805, by Abcam (1 mentions)
Axiocam hrm camera, by Zeiss (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Carl apotome axiovert 200 m fluorescence microscope, by Zeiss (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab191470, by Abcam (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Avantor (1 mentions)
Ab36057, by Cell Signaling Technology (1 mentions)
Sds page gels, by Avantor (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
6 protocols using ab133554
1
Protein Extraction and Immunoblotting
2
Immunofluorescence Staining of S100P and E-cadherin
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Cells were cultured to confluent monolayers on glass coverslips and fixed in ice-cold methanol for 20 min, except for S100P stained cells, which were fixed with 4% paraformaldehyde for 20 min. Following a 10 min PBS wash, cells fixed with paraformaldehyde were incubated in NH4Cl 50 mM for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 5 min, at room temperature. Cells were blocked with 3% BSA in PBS and stained with primary antibodies, rabbit anti-S100P (ab133554, Abcam, UK) and mouse anti-E-cadherin (610,182, BD Biosciences, USA), followed by a 1 h incubation in the dark with Alexa 488 or Alexa 594-conjugated secondary IgG (Invitrogen, Thermo Fisher Scientific, USA). Coverslips were mounted with Vectashield with DAPI (Vector Laboratories, USA) and images acquired on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope (× 20 and × 40 objectives; Carl Zeiss, Germany) with an Axiocam HRm camera. Images were processed with the Zeiss Axion Vision 4.8 software.
3
Western Blot Analysis of Endometrial Proteins
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Protein was extracted from endometrial tissue samples or cultured cells using RIPA buffer containing protease inhibitors (89,900, Thermo, USA). Equal amounts of 30 μg protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, United States). Then, the membranes were incubated with the following specific primary antibodies: S100P (ab133554, Abcam, UK, 1:1000), Bax (5023 T, Cell Signaling Technology, 1:1000), Bcl-2 (4223S, Cell Signaling Technology, 1:1000), HOXA10 (ab191470, Abcam, UK, 1:1000), GAPDH (5174, Cell Signaling Technology, 1:1000). Afterwards, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime Biotechnology, China, 1:1000). Finally, protein bands were detected by enhanced chemiluminescence (ECL) according to the manufacturer’s (Millipore) instructions.
4
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Total protein was isolated using RIPA buffer (Amresco, USA) containing protease inhibitor cocktail. Protein extract was separated on SDS-PAGE gels (Amresco, USA) followed by transfer to polyvinylidene fluoride membranes. The membranes were subsequently blocked in 5% defatted milk and incubated with primary antibody overnight at 4 °C. Following incubation with the appropriate secondary antibody conjugated to horseradish peroxidase, the blots were visualised using the enhanced chemiluminescence (FDbio-pico ECL, China). The following antibodies were included in western blotting: anti-S100P (Abcam #ab133554, UK), anti-SLC2A5 (Abcam #ab36057, UK), anti-E-cadherin (Cell Signaling Technology #3195, USA), anti-N-cadherin (Cell Signaling Technology #13116, USA), anti-Vimentin (Cell Signaling Technology, #5741, USA), anti-GAPDH (Proteintech #10494-1-AP, USA), anti- Mouse IgG (Cell Signaling Technology #5873, USA) and anti-Rabbit IgG (Cell Signaling Technology #14708, USA).
5
Protein Expression Analysis by Western Blot
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For Western blot analysis, cells were lysed on ice using a lysis buffer supplemented with a protease inhibitor cocktail (Sigma‐Aldrich, St. Louis, MO, USA). Total protein (30 μg) was separated by gel electrophoresis and transferred to PVDF membranes. The immunoreaction was carried out using primary antibodies against Trx‐1 (ab133524), S100P (ab133554), S100A4 (ab27957) (Abcam, ITK Diagnostics BV, The Netherlands); AKT (9272), phospho‐AKT (P‐AKT, 4060), β‐Actin (4970) (Cell Signaling Technology, Bioke, The Netherlands); E‐cadherin (610181), vimentin (550513) (BD Biosciences, San Jose, CA, USA); and fibronectin (sc18825) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
6
Immunohistochemical Analysis of S100P and β-hCG
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Paraffin slides were dewaxed with xylene for 20 min and 100%, 95%, and 75% ethanol in sequence for a total of 30 min and boiled in Antigen Retrieval Buffer (citrate buffer, pH 6.0, or Tris-EDTA buffer, pH 9.0) (ab93678 and ab93684, Abcam, USA) at 100°C for 15 minutes. The slides were blocked with 2% goat serum for 1 hour at room temperature and incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: S100P (ab133554, 1:100, Abcam, USA) and β-hCG (ab9582, 1:50, Abcam, USA). After washing with TBST buffer, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature (GK500710, Gene Tech, China). HRP-conjugated antibodies were detected with diaminobenzidine (DAB) (GK500710, Gene Tech, China) for 10 min. The nuclei were then stained with hematoxylin solution (ab220365, Abcam, USA) for an addition 5 min. Images were captured with an Axio Scope A1 (Zeiss, Germany).
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