Western Blot Analysis Optimization

Cells extraction, lysate quantification, SDS-Page and western blots were performed as described using stain-free gels [20 (link)]. Membranes were incubated with rabbit anti-β-catenin, mouse anti-GAPDH (GeneTex), goat anti-pGsk-3β (Ser9), rabbit anti-LaminB1, mouse anti-pCREB (Ser133), mouse anti-pMEK1/2, mouse anti-β-actin, rabbit anti-FAK, rat anti-pFAK (Y397, R&D Systems) rabbit anti-PI3K, goat anti-pPI3K (Tyr 508), goat anti-AKT1, rabbit anti-pAKT1 (Tyr308), rabbit anti-ERK1/2 (Cell Signaling Technology, Frankfurt am Main, Germany), rabbit anti pERK1/2 (Thr202/Tyr204, Cell Signaling Technology) as well as goat anti-rabbit, goat anti-rat, donkey anti-goat and anti-mouse IgG kappa binding protein IgG HRP-conjugated diluted in 1% BSA solution. If not indicated otherwise antibodies were purchased from Santa Cruz Biotechnology. Western blots were quantified via chemiluminescence using a Clarity Western ECL substrate chemiluminescence kit (BioRad). Besides the loading control β-Actin, we used stainfree total protein normalization. Membranes were photographed and quantified using ChemiDoc XRS+ imaging acquiring system (BioRad) and Image Lab software v. 6.0 (BioRad).

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Jakubzig B., Baltes F., Henze S., Schlesinger M, & Bendas G. (2018). Mechanisms of Matrix-Induced Chemoresistance of Breast Cancer Cells—Deciphering Novel Potential Targets for a Cell Sensitization. Cancers, 10(12), 495.

Publication 2018

mouse anti-GAPDH mouse anti-β-actin rabbit anti-ERK1/2 rabbit anti pERK1/2 (Thr202/Tyr204, Clarity Western ECL substrate chemiluminescence kit ChemiDoc XRS+ imaging acquiring system Image Lab software v. 6.0

Actin Akt1 Anti igg Antibodies Binding protein Cells Chemiluminescence Donkey Erk1 Gapdh Gels Goat Membranes Mouse Pi3k Protein Rabbit Sds page Stain Western blots β catenin

Corresponding Organization : University of Bonn

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

β-catenin
GAPDH
PGsk-3β (Ser9)
LaminB1
PCREB (Ser133)
PMEK1/2
β-actin
PFAK (Y397)
PPI3K (Tyr 508)
PAKT1 (Tyr308)
ERK1/2
PERK1/2 (Thr202/Tyr204)

control variables

None explicitly mentioned

positive controls

β-Actin

negative controls

None explicitly mentioned

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