ALA Pretreatment Modulates HS-Induced Stress Responses

Caco-2 cells pretreated with different ALA concentrations for 24 h and exposed to HS for 6 h (HSF1, Nrf2) or 24 h (HSP70, E-cadherin) were lysed using 50 µl RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA) containing protease inhibitors (Roche Applied Science, Penzberg, Germany). Lysates were normalized for protein content and WB analysis was conducted as described previously [17 (link)] using primary antibodies against HSF1 (1:1000; Cell Signaling, Danvers, MA, USA), HSP70 (1:1000; Enzo Life Sciences, Farmingdale, NY, USA) or E-cadherin (1:1000, eBioscience, San Diego, CA, USA), and β-actin antibody (1:4000; Cell Signaling) for equality of sample loading. For the detection of Nrf2, the nuclear protein extracts were separated using NE-PER Nuclear and Cytoplasmic Extraction Reagents based on the manufacturer’s instructions (Pierce, Rockford, IL, USA). Nuclear protein concentrations were measured, normalized, and the WB analysis was conducted using primary antibodies against Nrf2 (1:1000; Cell Signaling) and Lamin A (1:1000; Cell Signaling) for equality of loading. Digital images were obtained with ChemiDoc™ MP imager (Bio-Rad Laboratories Inc.) and signal intensities were quantified using the ImageJ 1.47 software.
The protein expression was normalized with β-actin or Lamin A (for nuclear proteins) and expressed as mean fold change in relation to the control group.