Vimentin Monoclonal Antibody (V9), Biotin

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For protein extraction, HK-2 cells were lysed on ice in lysis buffer (Tris 10mM à pH 7.5, NaCl 150mM, EDTA 1mM, EGTA 1mM, SDS 0.1%, NP40 1% and deoxycholate 1%) supplemented with a protease inhibitor cocktail (Complete, Mini, Roche).	Proteins were separated in a 4–15% SDS-PAGE and blotted on a nitrocellulose membrane (Amersham). Membranes were blocked with TBS-Tween 0.1% supplemented with milk 5% for 30 min. Then, membranes were incubated with primary antibodies (Anti E-Cadherin [1:200, Sc-7870 Santa Cruz], anti Fibronectin [1:500, F3648 Sigma], anti αSMA [1:500, Ab5964 AbCam], anti Vimentin [1:1000, MS-129-P Neomarkers], anti β-Actin (1:5000, A5441 Sigma)) overnight at 4°C followed by incubation with the peroxidase-conjugated secondary antibody (anti rabbit or anti mouse [1:10000, Bethyl]) for 1h.	Specific bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc XRS+ System (Bio-Rad). Protein bands were quantified by densitometry using Image Lab software (version 4.0.1, Bio-Rad) and results are expressed as ratio between target protein and β-Actin.

Upstream tips
For protein extraction, HK-2 cells were lysed on ice in lysis buffer (Tris 10mM à pH 7.5, NaCl 150mM, EDTA 1mM, EGTA 1mM, SDS 0.1%, NP40 1% and deoxycholate 1%) supplemented with a protease inhibitor cocktail (Complete, Mini, Roche).
Protocol tips
Proteins were separated in a 4–15% SDS-PAGE and blotted on a nitrocellulose membrane (Amersham). Membranes were blocked with TBS-Tween 0.1% supplemented with milk 5% for 30 min. Then, membranes were incubated with primary antibodies (Anti E-Cadherin [1:200, Sc-7870 Santa Cruz], anti Fibronectin [1:500, F3648 Sigma], anti αSMA [1:500, Ab5964 AbCam], anti Vimentin [1:1000, MS-129-P Neomarkers], anti β-Actin (1:5000, A5441 Sigma)) overnight at 4°C followed by incubation with the peroxidase-conjugated secondary antibody (anti rabbit or anti mouse [1:10000, Bethyl]) for 1h.
Downstream tips
Specific bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc XRS+ System (Bio-Rad). Protein bands were quantified by densitometry using Image Lab software (version 4.0.1, Bio-Rad) and results are expressed as ratio between target protein and β-Actin.

Publication protocol

For protein extraction, HK-2 cells were lysed on ice in lysis buffer (Tris 10mM à pH 7.5, NaCl 150mM, EDTA 1mM, EGTA 1mM, SDS 0.1%, NP40 1% and deoxycholate 1%) supplemented with a protease inhibitor cocktail (Complete, Mini, Roche). Proteins were separated in a 4–15% SDS-PAGE and blotted on a nitrocellulose membrane (Amersham). Membranes were blocked with TBS-Tween 0.1% supplemented with milk 5% for 30 min. Then, membranes were incubated with primary antibodies (Anti E-Cadherin [1:200, Sc-7870 Santa Cruz], anti Fibronectin [1:500, F3648 Sigma], anti αSMA [1:500, Ab5964 AbCam], anti Vimentin [1:1000, MS-129-P Neomarkers], anti β-Actin (1:5000, A5441 Sigma)) overnight at 4°C followed by incubation with the peroxidase-conjugated secondary antibody (anti rabbit or anti mouse [1:10000, Bethyl]) for 1h. Specific bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc XRS+ System (Bio-Rad). Protein bands were quantified by densitometry using Image Lab software (version 4.0.1, Bio-Rad) and results are expressed as ratio between target protein and β-Actin.

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