Protocol detail

Western Blot and ELISA Characterization

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Cells were lysed in TBST buffer as described previously [30 (link)]. The protein content was determined using the BCA™ Protein Assay Kit (Pierce). After transfer on PVDF membranes (BioRad) blots were blocked in 5% skim milk and incubated with antibodies (4 °C, overnight) directed against HSF-1 (ADI-SPA-901; Enzo Life Sciences Inc., Farmingdale, NY, USA), HSF-1 phospho S326 (pHSF-1) (ab76076; abcam), Hsp70 (ADI-SPA-810; Enzo Life Sciences; cmHsp70.1; multimmune GmbH), Hsp27 (ADI-SPA-800; Enzo Life Sciences), Akt (Cell Signaling Technology Europe, Frankfurt, Germany) and β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA). Horseradish-peroxidase (HRP)-conjugated anti-rabbit/anti-mouse antibodies (Promega, Fitchburg, WI, USA, W401B/W402B) were used as secondary antibodies. Immune complexes as detected by ECL detection system (GE Healthcare, Chicago, IL, USA) were imaged digitally (ChemiDoc Touch Imaging System, Biorad, Hercules, CA, USA). Hsp70 protein concentrations in cell lysates were quantified by ELISA (R&D systems, Minneaplis, MN, USA) following the manufacturer’s recommendations.

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Kühnel A., Schilling D., Combs S.E., Haller B., Schwab M, & Multhoff G. (2019). Radiosensitization of HSF-1 Knockdown Lung Cancer Cells by Low Concentrations of Hsp90 Inhibitor NVP-AUY922. Cells, 8(10), 1166.

Publication 2019

PVDF membranes ADI-SPA-901 ab76076 ADI-SPA-810 ADI-SPA-800 β-actin ECL detection system ChemiDoc Touch Imaging System ELISA

Actin Anti antibodies Antibodies Assay Buffer Cell Elisa Horseradish peroxidase Hsp27 Hsp70 Immune complexes Membranes Milk Mouse Promega Protein Pvdf Rabbit Touch

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Variable analysis

independent variables

Cells were lysed in TBST buffer

dependent variables

Protein content determined using the BCA™ Protein Assay Kit
Expression levels of HSF-1, phosphorylated HSF-1 (pHSF-1), Hsp70, Hsp27, Akt, and β-actin proteins measured by Western blot
Hsp70 protein concentrations in cell lysates quantified by ELISA

control variables

Blocking of blots in 5% skim milk
Incubation of blots with antibodies (4 °C, overnight)

controls

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

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