Sc 376023

Western blot analysis was performed as previously described [6 ]. Regular Western blot analysis was performed using LV lysates that were prepared using RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protein inhibitor mixture (Sigma-Aldrich)). In addition, Western blot analysis of protein aggregation was performed using LV detergent-soluble and -insoluble lysates. Briefly, detergent-soluble fractions of LV tissues were obtained using a 2% Triton X-100 lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 2% Triton X-100, and a protein inhibitor mixture (Sigma-Aldrich)). The insoluble fractions were further solubilized using the 2% Triton X-100 lysis buffer supplemented with 1% SDS. Primary antibodies included anti-LC3 (L7543, Sigma-Aldrich), anti-P62 (ab91526, abcam), anti-NQO1 (sc-376023, Santa Cruz Biotechnology, Inc.), Anti-ATG5 (A2859, Sigma-Aldrich), anti-ATG7 (A2856, Sigma-Aldrich), anti-LAMP1 (ab24170, Abcam), anticathepsin D (2284, CST), and anti-Ub (sc-8017, Santa Cruz Biotechnology, Inc.). Ubiquitinated proteins with molecular weights from the top to 35 kDa on the membranes of immunoblots were quantified

To evaluate HO-1, NQO1 and SOD expression, IEC-6 cells (2 × 10⁴ cells/well; 96-well plates) were treated with IS (31.2–250 μM) for 24 h.
Thereafter, the cells were collected, washed with PBS, and incubated with Fixing Solution for 20 min at 4 °C and successively for 30 min at 4 °C with Fix Perm Solution. Anti-HO-1 (sc-10789, Santa Cruz Biotechnology) or anti-NQO1 (sc-376023, Santa Cruz Biotechnology) or anti-SOD (sc-30080, Santa Cruz Biotechnology) antibody were subsequently added. The secondary antibody was added, for 30 min, in Fix Solution, and cell fluorescence was evaluated by FACSscan (Becton Dickinson) and elaborated with Cell Quest software, as previously reported [60 (link)].

We performed protein extraction and western blot analyses as described previously^{20 (link)}. The utilized antibodies included mouse antibody to NQO1 (sc-376023, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nrf2 (MAB3925, 1:500; R&D System), NFκB (p65) (sc-8008, 1:200; Santa Cruz Biotechnology), goat antibody to HO-1 (AF3169, 1:500; R&D System), β-actin (MAB8929, 1:500; R&D System), goat anti-mouse IgG (sc-2039, 1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and donkey anti-goat IgG (sc-2042, 1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The proteins were visualized using an enhanced chemiluminescence detection kit (Bio-Rad, USA) following the manufacturer’s protocol. Images were analyzed using the Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA). Protein bands intensity were normalized to β-actin, and the data expressed in terms of percent relative to controls.