Western Blot Analysis of Cellular Markers

Cells were washed with cold PBS, scraped into RIPA buffer and centrifuged. The cell lysates were subjected to 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) hybridization transfer membrane. The primary antibodies used were as follows: HA (Sc-7392, Santa Cruze), CD44 (15675-1-AP, ProteinTech group), Fibronectin (sc-8422, Santa Cruze), Vimentin (5741, Cell Signaling Technology), p21cip1 (sc-6246, Santa Cruze), p27kip2 (sc-1641, Santa Cruze) and β-actin (Sc-47778, Santa Cruze). Secondary staining and detection were carried out in accordance with standard protocols^{16 (link), 64 (link)}.

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Xu H., Yu S., Yuan X., Xiong J., Kuang D., Pestell R.G, & Wu K. (2017). DACH1 suppresses breast cancer as a negative regulator of CD44. Scientific Reports, 7, 4361.

Publication 2017

Fibronectin Vimentin p21cip1 β-actin

Actin Antibodies Buffer Cd44 Cell Cold Fibronectin Hybridization Membrane P21cip1 Polyvinylidene fluoride Ripa Sds page Vimentin

Corresponding Organization :

Other organizations : Tongji Hospital, Huazhong University of Science and Technology, Sidney Kimmel Cancer Center, Thomas Jefferson University

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Variable analysis

independent variables

Treatment with primary antibodies: HA, CD44, Fibronectin, Vimentin, p21cip1, p27kip2, and β-actin

dependent variables

Protein expression levels of HA, CD44, Fibronectin, Vimentin, p21cip1, p27kip2, and β-actin

control variables

Cell lysis protocol: Cells were washed with cold PBS, scraped into RIPA buffer and centrifuged
Protein separation: Cell lysates were subjected to 10% SDS-PAGE
Protein transfer: Proteins were transferred to a polyvinylidene fluoride (PVDF) hybridization transfer membrane
Secondary staining and detection: Carried out in accordance with standard protocols

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