Laemmli loading buffer

For Western blotting analysis, 10⁶ cells were lysed in Laemmli loading buffer (Sigma Aldrich). The cell extract was then sonicated and heated for 5 min at 95° C. Cell lysates were separated by SDS-PAGE using 9% Bis-Tris polyacrylamide gels. Proteins were transferred to Amersham Protran nitrocellulose membranes (0.20 μm-pore size; GE Healthcare, Little Chalfont, UK). Immunoblots were probed overnight at 4 °C in TBS-Tween (100 mmol·L⁻¹ Tris–HCl, 150 mmol·L⁻¹ NaCl and 0.1% Tween-20, pH 7.6) with 3% fat milk with one of the antibodies listed in Table S1. Membranes were washed and incubated for 1 h at 4 °C with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5000, GE Healthcare, Little Chalfont, UK). Membranes were washed three times for 5 min per wash with TBS-Tween, and bound antibodies were detected using ECL chemiluminescent substrate (Immobilon, Millipore, Billerica, MA, USA). Results were analyzed with GeneGnome XRQ (SYNGENE Ozyme, Cambridge, UK). Quantification was performed using GeneTools from syngene. SOX2 expression was normalized to GAPDH for each sample, and then the normalized SOX2 expression in the treated samples was expressed relative to DMSO (control) treatment.

Treated monolayers of T₈₄ cells were scraped from their inserts and homogenized in lysis buffer (130 mM glycine, 2% sodium dodecyl sulphate [SDS], 7.7% glycerol in 70 mM Tris‐HCl, pH 8.8) by repeated passage through a 26‐gauge needle. Samples, normalized for protein content, were mixed with an equal volume of 2 × laemmli loading buffer (1/1, v/v) (Sigma), boiled for 5 min, and loaded onto a 8% SDS‐tricine polyacrylamide gel. After electrophoresis, transfer to PVDF membranes (Millipore) was performed for 2 hr at 0.15 A in 0.05 M sodium borate solution, pH 9.0, with 20% methanol and 0.05% SDS. Immunoblotting was performed with antibodies against cleaved‐PARP (Catalogue #: 9,546; Cell Signalling Technology). cleaved‐PARP levels were quantified by densitometry (ImageQuantTLInk software) and normalized to β‐actin as a protein loading control (Abcam).