Quantifying Protein Levels: Western Blotting and Immunofluorescence

The protein levels were quantified by western blotting analysis of cell extracts using antibodies below: anti-GAPDH (1:2000, Santa Cruz, USA), anti-PN-1, anti-E-cadherin, anti-Vimentin, anti-Snail, anti-ZEB-1, anti-MMP9, anti-Oct4, anti-Sox2, anti-Nanog, anti-EGFR, anti-ERK1/2, anti-P-ERK1/2, anti-P-PKCδ, anti-PKCδ, anti-EGR1, anti-htrA1, and anti-EGF (1:1000, Abcam, USA). ImageJ software was used for protein bands analysis. Immunofluorescence assay was performed as described elsewhere^{15 (link)}, in which the monoclonal antibodies, including anti- PKCδ (1:100, Abcam) and anti-Vimentin (1:200, Abcam) antibodies were used. Cell images were captured using a laser scanning confocal microscopy (Olympus, Japan).

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Tang T., Zhu Q., Li X., Zhu G., Deng S., Wang Y., Ni L., Chen X., Zhang Y., Xia T., Zen K., Pan Y, & Jin L. (2019). Protease Nexin I is a feedback regulator of EGF/PKC/MAPK/EGR1 signaling in breast cancer cells metastasis and stemness. Cell Death & Disease, 10(9), 649.

Publication 2019

anti-GAPDH anti-E-cadherin anti-Vimentin anti-Snail anti-ZEB-1 anti-MMP9 anti-Oct4 anti-Sox2 anti-Nanog anti-EGFR anti-ERK1/2 anti-P-ERK1/2 anti-PKCδ anti-EGR1 anti-EGF laser scanning confocal microscopy

Antibodies Cadherin Cell Cell extracts Egfr Egr1 Erk1 Gapdh Htra1 Immunofluorescence assay Laser scanning confocal microscopy Mmp9 Monoclonal antibodies Oct4 Protein Snail Sox2 Vimentin Western blotting analysis

Corresponding Organization :

Other organizations : China Pharmaceutical University, Nanjing Medical University, Jiangsu Province Hospital, Nanjing University

Top 5 similar protocols

Variable analysis

independent variables

Anti-GAPDH (1:2000, Santa Cruz, USA)
Anti-PN-1
Anti-E-cadherin
Anti-Vimentin
Anti-Snail
Anti-ZEB-1
Anti-MMP9
Anti-Oct4
Anti-Sox2
Anti-Nanog
Anti-EGFR
Anti-ERK1/2
Anti-P-ERK1/2
Anti-P-PKCδ
Anti-PKCδ
Anti-EGR1
Anti-htrA1
Anti-EGF (1:1000, Abcam, USA)
Anti-PKCδ (1:100, Abcam)
Anti-Vimentin (1:200, Abcam)

dependent variables

Protein levels quantified by western blotting analysis
Immunofluorescence assay

control variables

Cell extracts

positive controls

Not specified

negative controls

Not specified

Annotations

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