N myc ncm 2 100

Antibodies for immunostaining and western blotting were used as per the manufacturer’s recommendations. The following antibodies were used within this study: β-tubulin (Cell Signalling Technology, Cat # 2146 (1:1000)), Cleaved Caspase-3 [Asp175] (Abcam, Cat # ab49899 (WB 1:500, IHC 1:1500)), CDKN2A/p19ARF (Abcam, Cat # ab80 (1:1000)), cMYC [Y69] (Abcam, Cat # ab32072 (WB 1:1000, IHC 1:500)), Cyclophilin B (Cell Signalling Technology, Cat # 43603 (1:1000)), GFAP [GA5] (Millipore, Cat # MAB3402 (1:1000)), GFP (Abcam, Cat # ab13970 (1:1000)), HSF1 (Abcam, Cat # ab61382 (1:1000)), HSP70 (Abcam, Cat # ab2787 (1:1000)), HSP90 [EPR16621] (Abcam, Cat # ab203085 (1:1000)), Ki67 [SP6] (Abcam, Cat # ab16667 (1:2000)), n-MYC [NCM II 100] (Abcam, Cat # ab16898 (1:250)), NPR3 (Abcam, Cat # ab97389 (1:250)), Olig-2 (Millipore, Cat # AB9610 (1:1000)), OTX2 (R&D Systems, Cat # AF1979 (1:500)), p21 (Abcam, Cat # ab109199 (1:1000)), Synaptophysin [SY38] (Millipore, Cat # MAB5258 (1:500)), TUJ1 (Covance, Cat # MMS-435P (1:500)), β-actin (Santa Cruz Biotechnology, Cat # sc-47778 (1:1000)), CDKN2A/p16INK4A (Abcam, Cat # ab211542 (1:1000)), anti-mouse IgG secondary HRP (VWR, Cat # NXA931 (1:1000)), anti-rabbit IgG secondary HRP (GE Healthcare, Cat # NA934 (1:1000)), and Lamin B1 (Abcam, Cat # ab16048 (1:1000)).

Cells and xenograft tumor samples were resuspended in high SDS-RIPA Buffer (50 mM Tris-HCl, pH 7.5, 150 mM Sodium Chloride, 1 % Triton X-100, 1 % sodium deoxycholate, 1 % SDS, 2 mM EDTA; Sigma Aldrich). Tissues were disrupted and homogenized with a TissueLyser II (Qiagen) for 2 × 2 min intervals at 30Hz. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Pierce). A total of 15–50 μg of protein extracts were loaded onto NuPAGE® Novex® 4–12 % Bis-Tris Protein Gels (Life Technologies) and subsequently transferred onto nitrocellulose membranes using the iBlot® Dry Blotting System (Life Technologies). The blots were developed using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific). Antibodies: S6-Ribosomal protein (5G10), Phospho-S6 Ribosomal protein (Ser240/244) (D68F8), Phospho-4E-BP1 (Thr37/46) (236B4), p44/42 MAPK (Erk1/2) (137 F5), and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) were purchased from Cell Signaling Technology. C-MYC (Y69) and N-MYC (NCM II 100) were purchased from Abcam. FLAG (M2) and β-actin (A2066) antibodies were purchased from Sigma Aldrich.