Quantitative Western Blot Analysis of Extracellular Matrix Proteins

Western blot analysis was performed using a standard protocol^{36 (link)}. Briefly, equal amounts of protein (30 μg) were separated via SDS-PAGE and electro-transferred onto polyvinylidene difluoride membranes (EMD Millipore). Membranes were probed with primary anti-TNC (ab108930, Abcam), anti-MMP2 (ab86607, Abcam), anti-MMP9 (ab76003, Abcam), anti-pan-Akt (4691 s, CST), anti-pAkt⁴⁷³ (4060 P, CST), anti-pAkt³⁰⁸ (sc-135650, Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (8457 S, CST) antibodies and then probed sequentially with secondary goat anti-mouse and anti-rabbit antibodies (7076P2, 7074P2, CST) and visualized using an enhanced chemiluminescence kit (Beyotime, Shanghai, China).

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Cai H.P., Wang J., Xi S.Y., Ni X.R., Chen Y.S., Yu Y.J., Cen Z.W., Yu Z.H., Chen F.R., Guo C.C., Zhang J., Ke C., Wang J, & Chen Z.P. (2019). Tenascin-c mediated vasculogenic mimicry formation via regulation of MMP2/MMP9 in glioma. Cell Death & Disease, 10(12), 879.

Publication 2019

ab108930 ab86607 ab76003 enhanced chemiluminescence kit

Actin Anti antibodies Antibodies Chemiluminescence Goat Membranes Mmp2 Mmp9 Mouse Polyvinylidene difluoride Protein Rabbit Sds page Western blot analysis

Corresponding Organization :

Other organizations : Sun Yat-sen University Cancer Center, Sun Yat-sen University

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Variable analysis

independent variables

Primary antibodies used for probing the membranes: anti-TNC, anti-MMP2, anti-MMP9, anti-pan-Akt, anti-pAkt^473, anti-pAkt^308, anti-β-actin

dependent variables

Protein expression levels of TNC, MMP2, MMP9, pan-Akt, pAkt^473, pAkt^308, and β-actin

control variables

Equal amounts of protein (30 μg) loaded onto the SDS-PAGE gel
Polyvinylidene difluoride membranes used for electro-transfer
Secondary antibodies (goat anti-mouse and anti-rabbit) used for visualization

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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