Goat anti rabbit igg h l peroxidase conjugated antibody

For lysing cells and to extract protein RIPA buffer was used, adding a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). For the SDS-PAGE assay, a total of 20 µg/sample was loaded in the acrylamide gel. Proteins were transferred from the gel to 0.2 µm pore-size nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were then blocked for 1 h using 5% BSA (PanReac AppliChem, ITW Reagents) in 1X TBS-T (0.1% Tween20, Bio-Rad). After membrane blocking, the following antibodies were used for blotting proteins: anti-HMGA1 (ab129153; Abcam), anti-PARP1 (51-66396R; BD Bioscience, Franklin Lakes, NJ, USA), anti-cleaved Caspase 3 (#9661; Cell signaling, Danvers, MA, USA), anti-phospho-S6 (#4858; Cell signaling), anti-S6 (#2217; Cell signaling) and anti-α-tubulin (Sigma-Aldrich, T9026). Membranes were consequently incubated with Rabbit Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich) or Goat Anti-Rabbit IgG H&L peroxidase-conjugated antibody (Abcam) secondary antibodies. For chemiluminescent detection, the HRP substrate was used. Image acquiring was performed using the ChemiDoc Imaging System (Bio-Rad). ImageJ was used to quantify protein expression levels.

Cell lysis and protein extraction was carried out using the RIPA buffer [1 M Tris–HCl pH 8 (PanReac AppliChem, ITW Reagents), 0.5 M EDTA (Thermo Fisher Scientific), Triton™ X-100 (Sigma-Aldrich), 10% sodium deoxycholate (Sigma-Aldrich), 10% SDS (Sigma-Aldrich) and 3 M NaCl (Thermo Fisher Scientific)], supplemented with protease and phosphatase inhibition cocktails (Sigma-Aldrich). 20 µg protein sample were separated by SDS-PAGE, then transferred to 0.2 µm pore-size nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h using 5% BSA (PanReac AppliChem, ITW Reagents), in 1X TBS-T (0.1% Tween20, Bio-Rad). Subsequently, the following antibodies were incubated in the same buffer for 16 h at 4 ºC: ISG15 (Santa Cruz Biotechnology, sc-166755), α-tubulin (Sigma-Aldrich, T9026). Membranes were washed with 1X TBS-T, then incubated with Rabbit Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich) or Goat Anti-Rabbit IgG H&L peroxidase-conjugated antibody (Abcam, Cambridge, UK). HRP substrate was used for chemiluminescent detection and image acquisition was performed using a Chemidoc Imaging System (Bio-Rad). ISG15 expression was relativised against INT-SFT values.