Tris hcl sds page precast gels

HSJD-DIPG007 and RA055 cells were plated at a cell density of 0.5–1 million cells and allowed to grow as spheroids. Subsequently cells were treated with Dox, NAs-HSA, and NAs-HSA-Dox at indicated doses for 24 h. Following treatments cells were washed with PBS and proteins were extracted using the RIPA Buffer according to manufacturer protocol, (Sigma-Aldrich). Protein content was subsequently quantified with the BCA Protein Acid Assay Kit (Pierce, ThermoFisher Scientific, Waltham, MA, USA). Proteins were resolved on 12–20% Tris-HCl SDS-PAGE precast gels (Bio-Rad, Gladesville, NSW, Australia) and blotted as described by manufacturer’s instructions (Bio-Rad). DIPG cell lysates were analysed with the following antibodies overnight at 4 °C; cleaved caspase 3 (1:1000, #9664S), cleaved parp (1:1000, #5625S), P-H2AX (1:1000, #9718S), GAPDH (1:2000, #2118), beta-actin (1:2000, #4970) while anti-rabbit HRP-linked monoclonal antibody was used as a secondary antibody for 2 h at room temperature (1:2000, #7074). All antibodies were purchased from Cell Signalling (Danvers, MA, USA).

Whole-cell extracts were obtained using RIPA Buffer according to the manufacturer’s protocol, (SigmaAldrich) and protein quantified by BCA Protein Acid Assay Kit (Pierce). Proteins were resolved on 12–20% Tris-HCl SDS-PAGE precast gels (Bio-Rad) and blotted (Bio-Rad) as described by the manufacturer’s instructions. Cell lysates were analyzed with the following antibodies ODC1 (1:500, Cat No: ab97395), SLC3A2 (1:1000, Cat No: VPA00372), cleaved – PARP (1:1000, Cat No: 9541S), Caspase 8 (1:1000, Cat No: 4790S), Phospho-Histone H2A.X (1:1000, Cat No: 9718), p-m-TOR (1:1000, Cat No: 2971S), m-TOR (1:1000, Cat No: 2983), p-4EBP1 (1:1000, Cat No: 236B4), 4EBP1 (1:1000, 9644S), Actin (1:1000, Cat No: D6A8), GAPDH (1:5000, Cat No:97166), Anti-Rabbit IgG HRP-linked antibody #7074, Anti-Mouse IgG HRP-linked antibody #7076. Original scans for all western blots are provided in Supplementary Figs. 28–35.