Anti hsp70 antibody

All cell samples (control and Cd-treated) were harvested, centrifuged and pellets were obtained by further centrifugation in PBS containing protease inhibitors. Homogenates were obtained by passing the cell samples though a syringe needle (22–23 ga diameter) and by incubation on ice for 15 min. The samples were then sonicated (10–15 s on ice) and centrifuged for the separation of the proteins of interest. The supernatants were stored at −80 °C in sample buffer until use. In this case, 30 µg of proteins were used and separated on 7% gel (Invitrogen Thermo Fisher Scientific) and transferred onto nitrocellulose membrane. A mouse monoclonal anti-Hsp70 antibody (Enzo Life Sciences, Euroclone, Pero-MI, Italy) was used for protein visualization. The same cell extracts were incubated with anti-vinculin antibody (Enzo Life Sciences, Switzerland) for correct protein loading visualization.
Densitometric analysis by Scion Image software (Scion Corp. Fredrick, MD, USA) provided the quantification of proteins expression (hsp70) in Cd-treated samples compared to controls.

Protein from treated and untreated cell lysates was estimated using the BCA protein estimation assay (Thermo Scientific). For Western blotting, anti-HSP70 antibody (Enzo life sciences), and anti-Sp1 antibodies (Cell Signaling) were used to check for levels of different proteins in the lysates. Actin was used as loading control. Actin antibody was obtained from Cell Signaling Technology.