Western blot analysis was performed as described previously36 (link). The primary antibodies were as follows: anti-p27 (ab193379, Abcam), anti-phosphorylated Rb (Ser795) (#9301, Cell Signaling Technology), anti-E2F1 (#3742, Cell Signaling Technology), anti-p21 (#2947, Cell Signaling Technology), anti-caspase-3 antibody (ab32351, Epitomics), anti-FHL2 (ab12327, Abcam), anti-β-catenin (#8480, Cell Signaling Technology), anti-phosphorylated β-catenin (Ser33/37/Thr41) (#9561, Cell Signaling Technology), anti-cyclin D1 (60186-lg, Proteintech), anti-p53 (10442-1-AP, Proteintech), anti-PUMA (ab33906, Epitomics), anti-E-cadherin (20874-1-AP, Proteintech), anti-N-cadherin (22018-1-AP, Proteintech), and anti-GAPDH (#2118, Cell Signaling Technology).
Ab12327
Manufactured by Abcam
Sourced in United States
Ab12327 is a laboratory equipment product from Abcam. It serves as a core function to support research activities, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab32351, by Abcam (2 mentions)
Ab193379, by Abcam (1 mentions)
Anti e2f1, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti n cadherin 22018 1 ap, by Proteintech (1 mentions)
Anti phosphorylated rb ser795, by Cell Signaling Technology (1 mentions)
Anti phosphorylated β catenin ser33 37 thr41, by Cell Signaling Technology (1 mentions)
Anti cyclin d1 60186 lg, by Proteintech (1 mentions)
Anti e cadherin 20874 1 ap, by Proteintech (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti p53, by Proteintech (1 mentions)
Ab15148, by Abcam (1 mentions)
Ab6302, by Abcam (1 mentions)
Hre luciferase, by Addgene (1 mentions)
On targetplus smartpool, by Thermo Fisher Scientific (1 mentions)
Sc 23900, by Santa Cruz Biotechnology (1 mentions)
Ab202030, by Abcam (1 mentions)
Ab193555, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
4 protocols using ab12327
1
Western Blot Analysis of Cell Signaling Proteins
2
Regulation of MRTFA-CTNNB1 Signaling
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The p3Xflag‐MKL1‐N200 expression vector was kindly provided by Professor R. Prywes (Colombia University, New York, NY, USA). It encodes a constitutively nuclear and active form of MRTFA (MRTFA‐CA). pcDNA3‐S33Y CTNNB1 (#19286), M50 Super 8x TOPFlash (TCF4‐Luc #12456) and HRE‐luciferase (#26731) were purchased from Addgene. Human MRTFA (ON‐TARGETplus® SMARTpool®) and esiRNA human HIF1A were purchased from Thermo Scientific and Sigma respectively. Antibodies against the following proteins were used: CTNNB1 (ab6302 Abcam), CDH1 (ab15148 Abcam), ERK (extracellular signal‐regulated kinase 1); K‐23 ESR1 (HC‐20, SC‐543, Santa Cruz Biotechnology), FHL2 (ab12327; Abcam), HIF1A (610958 BD Biosciences), MRTFA (ab113264 Abcam) and phalloidin‐iFluor 594 (lab176757; Abcam).
3
Immunohistochemical Analysis of Tumor Samples
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Paraffin sections (5 μm) from tumor tissue samples were deparaffinized in 100% xylene and rehydrated with a decreasing ethanol series and then water, according to standard protocols. Heat-induced antigen retrieval was performed in 0.01 mol/L sodium citrate buffer (pH 6.0) for 10 min at 100 °C. Endogenous peroxidase activity and non-specific antigens were blocked, followed by incubation with anti-FHL2 antibody (ab12327, Abcam), anti-Ki67 antibody (sc-23900, Santa Cruz), or anti-caspase-3 antibody (ab32351, Epitomics), overnight at 4 °C. After washing, the sections were incubated with Poly-HRP-Conjugated Anti-Rabbit IgG or Poly-HRP-Conjugated Anti-Mouse IgG (Sangon Biotech) for 45 min at room temperature. The sections were visualized with 3,3′-diaminobenzidine (DAB), counterstained with hematoxylin, mounted in neutral gum and analyzed using a bright field microscope.
4
Comprehensive Protein Analysis by Western Blot
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Western blot analysis was conducted as previously described [27 (link)]. The primary antibodies against proliferating cell nuclear antigen (PCNA; ab29; 1:1000), total-caspase 3 (ab13847; 1:500), cleaved-caspase-3 (C-caspase 3; ab13847; 1:1000), N-cadherin (ab202030; 1:1000), Vimentin (ab193555; 1:1000), E-cadherin (ab1416; 1:1000), FHL2 (ab12327; 1:1000) and GAPDH (ab8245; 1:1000), and the secondary antibodies, horseradish peroxidase (HRP) conjugated goat anti-rabbit (ab205718; 1:5000) and goat anti-mouse (ab205719; 1:5000), were purchased from Abcam (Cambridge, MA, USA).
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