Anti myc 9e10

Expression vectors for indicated proteins were transiently transfected into human 293 T embryonic kidney cells (ATCC CRL-3216) by the calcium phosphate coprecipitation method^{46 (link)}. Approximately 40 h later, cells were lysed^{47 (link)} and immunoprecipitations performed as previously described^{48 (link)} with either anti-Flag M2 (Sigma-Aldrich F1804) or anti-HA 12CA5 (Santa Cruz Biotechnology sc-57592) mouse monoclonal antibodies. Immunoprecipitates as well as inputs were boiled in Laemmli sample buffer^{49 (link)} and subjected to polyacrylamide gel electrophoresis^{50 (link)}. Separated proteins were transferred to polyvinylidene difluoride membranes^{51 (link)} and challenged with anti-Flag (Sigma-Aldrich F7425) or anti-Myc (Santa Cruz Biotechnology sc-789) rabbit polyclonal antibodies or anti-HA 12CA5 or anti-Myc 9E10 (Sigma-Aldrich M4439) mouse monoclonal antibodies^{52 (link)}. This was followed by incubation with horseradish peroxidase-coupled secondary antibodies^{53 (link)} and signal detection through enhanced chemiluminescence^{54 (link)}.

Anti-Rbfox1(A2BP1) was produced by ourselves as previously described28 (link). The following mouse monoclonal antibodies were used; anti-Tau-1 (MAB3420; Chemicon International, Temecula, CA), anti-Myc 9E10 and anti-MAP2 (M4403; Sigma-Aldrich, St Louis, MO). Polyclonal rabbit antibodies used were anti-GFP (#598; MBL, Nagoya, Japan), anti-RFP (#600-401-379; Rockland Immunochemicals, Gilbertsville, PA), anti-Rbfox2 (Fox2) (A300-864A-T, Bethyl Laboratories, Montgomery, TX) and anti-Sept1129 (link). anti-GFP (GFP-1020; Chicken) was purchased from AVES Labs (Tigard, OR).

Cells were lysed in cell lysis solution (Pierce) on ice for 1 h and centrifuged at 12,000 rpm for 5 min to remove cell debris. The supernatants were subjected to SDS-PAGE. Proteins were then transferred to a 0.45-μm polyvinylidene difluoride (PVDF) membrane and labeled with specific antibodies. The antibodies used in this subject were as follows: anti-MYC (9E10, 1:1,000), anti-HA (H9658, 1:1,000), anti-FLAG (F1804, 1:1,000), and anti-beta-actin (A2066, 1:1,000) were from Sigma; anti-SAMHD1 (ab67820, 1:1,000), anti-NS3 (ab65407, 1:1,000), and anti-Core (ab2740, 1:1,000) were from Abcam; anti-SREBP-1 (39940, 1:1,000) was from Active Motif; HRP-conjugated goat anti-mouse (ZSGB-Bio, catalog ZB2305, 1:5,000) and goat anti-rabbit (ZSGB-Bio, catalog ZB2301, 1:5,000) were secondary antibodies.

Human embryonic kidney (HEK)−293T cells were cultured in the Dulbecco's modified eagle medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Thermo Fisher Scientific). Human colorectal adenocarcinoma SW480 cells were cultured in the Roswell Park Memorial Institute-1640 medium (Sigma-Aldrich) supplemented with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. Cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C. Lipofectamine 3000 transfection reagent was purchased from Thermo Fisher Scientific. GENE-fect was purchased from TransLab. Cycloheximide was purchased from Sigma-Aldrich. Protease and phosphatase inhibitor cocktails were purchased from GenDEPOT. The IP lysis buffer and Dynabeads protein G were purchased from Thermo Fisher Scientific. The anti-Myc (9E10)-HRP antibody was purchased from Abcam, and the anti-FLAG (M2)-HRP, anti-HA-HRP (3F10), anti-FLAG-M2, anti-Myc (9E10), and β-actin-HRP antibodies were purchased from Sigma-Aldrich.

A polyclonal antibody against ASPH was raised in our institution using the synthetic peptide antigen of 12 amino-acid residues around the Fe²⁺-binding domain of ASPH. The antibodies against mitochondrial biomarkers heat shock protein 60 (ab46798) and voltage-dependent anion channel (ab154856), endoplasmic reticulum biomarkers calnexin (ab195198), green fluorescent protein (ab6556), H2AX (ab11175), POLG (ab207558) and mtTFA (ab176558) were purchased from Abcam (Cambridge, UK). The mouse monoclonal anti-myc (9E10) and HA antibodies (12CA5) were purchased from Sigma-Aldrich (St Louis, MO, USA) and Roche (Basel, Switzerland), respectively. Additional information on secondary antibodies is detailed in the Supplementary Information.

Mouse anti-ubiquitin P4D1 and rabbit anti-GST Z-5 were from Santa Cruz. The mouse monoclonal (mAb) antibody anti-myc 9E10, and anti-tubulin TUB2.1 were from Sigma, mAb anti-P23 JJ3 from Abcam, mAb anti-actin C4 from Millipore and mAb anti-HA HA.11 from Covance. Horse-radish peroxidase-conjugated donkey anti-rabbit (GE healthcare) and goat anti-mouse antibodies (Thermoscientific) were used as secondary antibodies.