Anti β actin peroxidase ac 15

Total protein was extracted from NCI-N87 and AGS, after transfection with either PBS blank control, negative control scrambled circRNA, or scRNA21, at four different treatment time intervals: 1 hr, 8 hr, 24 hr, and 5 days. Mouse anti-Daxx (H-7; Santa Cruz Biotechnology) and anti-β-actin peroxidase (AC-15; Sigma-Aldrich), horseradish-peroxidase-conjugated goat anti-mouse IgG (Invitrogen), and ECL Western Blotting detection kits (Amersham-Pharmacia Biotech) were used.

Western blotting was performed as previously described¹⁵ (link) and according to standard protocols. Briefly, cells of at least three independent biological replicates were used and treated, as previously indicated, and lysed using 1x cytoplasmic extract buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.075% NP40). After incubation and centrifugation, the cytoplasmatic fraction was collected and nuclear proteins were obtained using nuclear extract buffer (20 mM Tris Cl, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF, 25% glycerol); Western Blotting and detection of the proteins of interest (POI) was performed using specific antibodies against COX-2 (12282S) and Caspase 3 (9662S) purchased from Cell Signaling (Leiden, The Netherlands), cleaved Caspase-3 (ab90437) purchased from abcam (Cambridge, UK) and anti β-Actin-Peroxidase (AC-15) purchased from Sigma-Aldrich (Darmstadt, Germany). Acrylamide gels for detection of POI and the housekeeping protein β-actin were run separately with same amount of protein lysates to avoid multiple use of membranes and thereby occurrence of unspecific bands. β-actin was used as a housekeeping protein due to its most stable and unchanged expression within the DIPG and HGG cell lines studied with and without treatment.