Ab9484

Western blot analyses of SOX3, SOX1 and SOX2 expression during RA induction were performed on whole cell lysates (WCL) extracted from uninduced NT2/D1 cells and cells induced with RA for 2, 4 and 7 days. For the analyses of SOX3 expression during treatments with 5-azaC WCL were isolated from NT2/D1 cells and cells treated with 5-azaC. WCL were isolated and western blots were performed as previously described [15 (link)] using anti-SOX3 (Abcam, ab42471), anti-SOX1 (Abcam, ab109290), anti-SOX2 (Active Motif, 39823), anti-α-tubulin (Calbiochem, DM1A) and anti- GAPDH (Abcam, ab9484), anti-caspase-3 (Cell Signaling, 9662), followed by the incubation with horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies (Amersham Biosciences, NJ, USA, diluted 1:10000). Immunoreactive bands were detected by Immobilion Western Chemiluminescence substrate HRP (Millipore, MA, USA). Density of protein bands on blots were quantified using ImageJ software (https://imagej.nih.gov/). Data from at least 3 independent experiments were normalized by the amount of α-tubulin and presented relative to the corresponding value for untreated cells.

We applied the PARIS Kit (Life Technologies) to separate nuclear and cytoplasmic fractions in accordance with the instructions of manufacturers. The concentration of total protein was detected with the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). We separated the extracted proteins with SDS-PAGE, which were transferred to PVDF membranes. 5% non-fat milk was used to block the membrane, which was incubated with primary antibodies against NOTCH1 (ab65297, Abcam, Cambridge, UK, 1:800), PAX8 (ab53490, Abcam, 1:1000), Ub (sc-166,553, Santa Cruz Biotechnology, 1:500), Histone H3.1 (ab1791, Abcam, 1:500), HA (Cat#2367, Cell Signaling Technology, NY, USA, 1:2000), and anti-GAPDH (ab9484, CellSignaling, Danvers, MA, 1:1000).

Western blot analysis was performed as previously described [6 (link)]. For Western blot analysis of phospho-p53, we added phosphatase inhibitors in lysis buffer (PhosSTOP, Roche). The primary antibodies were: rabbit polyclonal antibody against SOX14 (Abcam, Cambridge, UK, ab149047, diluted 1:400), mouse monoclonal anti-α-Tubulin (Calbiochem, MA, USA, CP06, diluted 1:30000), mouse monoclonal antibody against GAPDH (Abcam, Cambridge, UK, ab9484, diluted 1:5000), mouse monoclonal antibody against cleaved PARP (Cell Signaling, Technology, Danvers, MA, USA, Asp214, diluted 1:1000), mouse monoclonal antibody against p53 (DO-1) (Santa Cruz Biotechnology, Texas, USA, sc-126X, diluted 1:1000), rabbit monoclonal antibody against p21^Waf1/Cip1 (Cell Signaling, Technology, Danvers, MA, USA, 2947, diluted 1:1000) and rabbit antibody against phospho-p53 (Cell Signaling, Technology, Danvers, MA, USA, diluted 1:1000). Secondary antibodies used for Western blot were: horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG (Amersham Biosciences, NJ, USA, diluted 1:10000 or Active motif, La Hulpe, Belgium, 1:5000). Immunoreactive bands were detected by chemiluminescence (Immobilion substrate, Millipore, MA, USA). The density of protein bands on Western blots was quantified using ImageJ software.