Thermo scientific halt protease inhibitor cocktail

Total protein was extracted from tissue samples using RIPA lysis buffer (Beyotime Biotechnology) supplemented with Thermo Scientific Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Protease inhibitors and phosphatase inhibitors (1:100) were added during protein extraction (MedChemExpress), and a Pierce BCA Protein Analysis Kit (Thermo Fisher Scientific) was used to measure protein concentrations. Protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk and incubated with the respective primary antibodies overnight at 4°C. The samples were incubated with horseradish peroxidase‐conjugated secondary antibodies (1:5000 dilution; Cell Signaling Technology) for 1 h at room temperature, and an iBright FL1500 imaging system (Invitrogen) and Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Invitrogen) were used to detect and analyse protein expression levels. Antibody information was provided for anti‐CD44 (60224‐1‐Ig; Proteintech), anti‐MYCN (ab16898; Abcam), and rabbit anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (WB: 1/1000; Cell Signaling Technology).

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Biotechnology, China) with 1× complete ULTRA (Roche, USA); protein was extracted from bone marrow samples using an Invitrogen™ TRIzol™ Kit (Thermo Fisher, USA) with the Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher, USA) according to the manufacturer’s instructions.

Total protein was extracted from cell samples using RIPA lysis buffer (Beyotime Biotechnology) supplemented with Thermo Scientific Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Protein extracts were resolved by SDS-PAGE and then electrophoretically transferred onto a PVDF membrane (EMD Millipore). The membrane was incubated for 2 h in blocking buffer (1 × TBST containing 5% non-fat milk), and then was incubated at 4℃ overnight with following primary antibodies: MIAC (abmart, 1:1000), AQP2 (abcam, ab199975), EREG (Cell Signaling, 12,048), EGFR (Proteintech, 18,986–1-AP), Phospho-EGFR (Abmart, T55232), mTOR(Abmart, T55306), p-mTOR (Abmart, T56571), Akt (Abmart, T55561), Phospho-Akt (Abmart, T40067), ERK1/2 (Abmart, T40071), Phospho-ERK1/2 (Abmart, TP56192), GAPDH (Proteintech, 60,004–1-Ig), HA (Abmart, M20003S). The immunocomplexes were subsequently incubated with the HRP-conjugated secondary antibodies, and detected with the Western Blot Detection kit (Beyotime Biotechnology) and TANON-5200 system (Tanon, Shanghai, P.R. China). The densitometric ratio of protein bands was calculated by ImageJ program.

Proteins extracted from primary patient cells or cell lines were resolved by 7.5%, 10%, or 12% Bis-Tris polyacrylamide gels and were transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% BSA for 1 h and probed with the appropriate antibody overnight at 4 °C and then were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Membranes were visualized with an enhanced chemiluminescence detection system. Detail information of antibody was mentioned in Additional file 1: Table S5.
For RIP assays, FLAG- or HA-tagged fusion proteins were used. In the RIP experiment with FLAG-DOT1L (N- terminal) or HA-DOT1L (N- terminal) truncated mutants in 293T cells, anti-FLAG (Sigma, USA) or anti-HA (Sigma, USA) was used along with an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (17-701) (Merck Millipore, Germany) according to the manufacturer’s instructions. All proteins for RIP were lysed with cell lysis buffer supplemented with Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher, USA). Finally, all samples were suspended in 5× loading buffer and then denatured for 5 min at 100 °C, separated via SDS-PAGE, transferred to PVDF membranes, and blotted.