Protein Expression Analysis by Western Blot

An equal amount of proteins from whole-cell lysates were separated by 4–12% SDS-PAGE and were transferred to a nitrocellulose membrane. Blots were blocked for 1 h with 5% non-fat dry milk and then incubated over night with the following primary antibodies: anti-CA-IX (R&D), anti-N-cadherin, anti-β-catenin and anti-Vimentin (CST-9782), anti-pro-Caspase-3, anti-Cleaved-Caspase-3, anti-AKT (CST-9272), anti-pAKT (CST-9271), anti-ERK (CST-9102), anti-pERK (CST-9101), anti-MMP-2 (CST-33437), anti-CD44 (Abcam), anti-GADPH (G8795) and anti-Actin (A4700). After washing with 0.1% Tween-20 in PBS, the filters were incubated with their respective secondary antibodies for 1 h and analyzed using the ECL system. Densitometric analyses were performed on at least two different expositions to assure the linearity of each acquisition using ImageJ software (v1.46r) [23 (link)].

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Sarnella A., Ferrara Y., Albanese S., Omodei D., Cerchia L., De Simone G., Supuran C.T, & Zannetti A. (2023). Inhibition of Bone Marrow-Mesenchymal Stem Cell-Induced Carbonic Anhydrase IX Potentiates Chemotherapy Efficacy in Triple-Negative Breast Cancer Cells. Cells, 12(2), 298.

Publication 2023

anti-CA-IX anti-N-cadherin anti-pro-Caspase-3 anti-AKT anti-pAKT anti-ERK anti-pERK anti-MMP-2 anti-CD44 anti-GADPH anti-Actin

Actin Antibodies Ca ix Cadherin Caspase 3 Cd44 Cell Densitometric Membrane Milk Mmp 2 Nitrocellulose Pro caspase 3 Proteins Sds page Tween 20 Vimentin β catenin

Corresponding Organization : Institute of Biostructure and Bioimaging

Other organizations : Institute for Experimental Endocrinology and Oncology, University of Florence

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Variable analysis

independent variables

Amount of proteins from whole-cell lysates

dependent variables

Protein expression levels of CA-IX, N-cadherin, β-catenin, Vimentin, pro-Caspase-3, Cleaved-Caspase-3, AKT, pAKT, ERK, pERK, MMP-2, CD44, GADPH, and Actin

control variables

4-12% SDS-PAGE
Transfer of separated proteins to nitrocellulose membrane
Blocking of blots for 1 h with 5% non-fat dry milk
Incubation with primary antibodies overnight
Washing with 0.1% Tween-20 in PBS
Incubation with secondary antibodies for 1 h
Analysis using the ECL system
Densitometric analyses on at least two different expositions to assure linearity of each acquisition using ImageJ software

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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