The concentrations of HSP70 and HSP90 (in nanograms per microgram of total protein)
were determined using immunoblotting. Samples were prepared according to Tunnah et al. (2016 (link)) and
compared with a commercially available standard (recombinant rat HSP70/72, SPP
758; human native HSP90, SPP770; Enzo Life Sciences) to quantify the specific HSP in
each sample. For the primary antibody rabbit anti-HSP70/HSC70 (1:5000 dilution
of AS05-083A; Agrisera), we used a horseradish peroxidase-tagged goat anti-rabbit
secondary antibody to visualize at 1:10 000 dilution (SAB-300; Enzo Life
Sciences). For the HSP90 primary antibody (mouse anti-Hsp90; 1:2500 dilution of
SMC-107; StressMarq Biosciences Inc.), we used a horseradish peroxidase-tagged goat
anti-mouse secondary antibody at 1:5000 dilution (ab5870; Abcam), Cambridge, UK.
Protein bands were visualized in Lumigen ECL Ultra (TMA-6) reagents (Southfield, MI,
USA) and imaged using a Molecular Imager VersaDoc™MP 400 System (BioRad,
Berkley, CA, USA) and Quantity One 1-D Analysis software. Image
Lab® software (BioRad) was used for quantifying the band density
in each sample.
were determined using immunoblotting. Samples were prepared according to Tunnah et al. (2016 (link)) and
compared with a commercially available standard (recombinant rat HSP70/72, SPP
758; human native HSP90, SPP770; Enzo Life Sciences) to quantify the specific HSP in
each sample. For the primary antibody rabbit anti-HSP70/HSC70 (1:5000 dilution
of AS05-083A; Agrisera), we used a horseradish peroxidase-tagged goat anti-rabbit
secondary antibody to visualize at 1:10 000 dilution (SAB-300; Enzo Life
Sciences). For the HSP90 primary antibody (mouse anti-Hsp90; 1:2500 dilution of
SMC-107; StressMarq Biosciences Inc.), we used a horseradish peroxidase-tagged goat
anti-mouse secondary antibody at 1:5000 dilution (ab5870; Abcam), Cambridge, UK.
Protein bands were visualized in Lumigen ECL Ultra (TMA-6) reagents (Southfield, MI,
USA) and imaged using a Molecular Imager VersaDoc™MP 400 System (BioRad,
Berkley, CA, USA) and Quantity One 1-D Analysis software. Image
Lab® software (BioRad) was used for quantifying the band density
in each sample.