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4 protocols using 14c ser

1

In Vitro Protein Synthesis Assay

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L-Thr, L-Ser, dithiothreitol (DTT), ribonucleoside triphosphate (NTP), guanosine monophosphate (GMP), tetrasodium pyrophosphate, pyrophosphatase (PPiase), Tris-base, MgCl2, NaCl, activated charcoal, horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Sigma (St Louis, MO, USA). [14C]Thr, [14C]Ser and [α-32P]ATP were obtained from Perkin Elmer Inc. (Waltham, MA, USA). KOD-plus mutagenesis kits, ReverTra Ace quantitative polymerase chain reaction (qPCR) RT Kit and SYBR Green Realtime PCR Master Mix were obtained from TOYOBO (Osaka, Japan). Dynabeads protein G, Lipofectamine 2000 transfection reagent, and the protein synthesis kit (1-Step Human Coupled IVT Kit) were obtained from Thermo Scientific (Waltham, MA, USA). Qproteome nuclear protein kits and Ni2+-NTA Superflow resin were purchased from Qiagen Inc. (Hilden, Germany). Polyethyleneimine cellulose plates were purchased from Merck (Darmstadt, Germany).
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2

Protein Expression and Purification Protocol

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T4 DNA ligase and Lipo8000 transfection reagent were purchased from Beyotime Biotechnology (Shanghai, China). 2 × Taq Plus Master Mix (Dye Plus) and T4 polynucleotide kinase was purchased from Vazyme Biotechnology (Nanjing, China). The KOD-Plus Mutagenesis Kit and KOD-Plus Kit were from TOYOBO (Osaka, Japan). Ni2+-NTA Superflow resin was purchased from Qiagen Inc. (Hilden, Germany). Dynabeads Protein G, 4,6-diamidino-2-phenylindole (DAPI), MitoTracker, Pierce Silver Stain kit, and Alexa Fluor 488-conjugated secondary antibody were obtained from Thermo Scientific (Waltham, MA, USA). [14C]Thr, [14C]Ser and [14C]Tyr were obtained from Perkin Elmer Inc. (Waltham, MA, USA). PrimeScript RT Master Mix and PrimeSTAR Max DNA Polymerase were obtained from TaKaRa (Kyoto, Japan). Trelief Prestained Protein Ladder and oligonucleotide primers were obtained from Tsingke (Shanghai, China). Competent E. coli BL21(DE3) were purchased from Weidi Biotechnology (Shanghai, China). Serum-free Cell Cryopreservation Medium was obtained from Epizyme Biomedical Technology (Shanghai, China). Anti-c-Myc magnetic beads, kanamycin sulfate and ampicillin sodium were purchased from MedChemExpress (MCE, New Jersey, USA).
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3

Antibody and Reagent Purchasing Protocol

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Anti-FLAG (F7425), anti-Myc (M4439) and anti-GAPDH (G8795) antibodies were purchased from Sigma (St. Louis, MO, USA). Anti-His6 (AE003) was purchased from Abclonal (Shanghai, China). [3H] SAM, [3H] Arg, [14C] Thr and [14C] Ser were obtained from Perkin Elmer Inc. (Waltham, MA, USA). Dynabeads Protein G, MitoTracker and Lipofectamine 2000 transfection reagent were obtained from Thermo Scientific (Waltham, MA, USA). Primers were synthesized in Biosune (Shanghai, China), and DNA sequencing was performed by Tsingke (Shanghai, China).
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4

Enzymatic Synthesis of ac4C RNA

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[14C]Acetyl-CoA and [14C]Ser were purchased from PerkinElmer (Waltham, MA, USA). KOD-Plus mutagenesis kits were purchased from TOYOBO (Osaka, Japan). Yeast was transformed using the Yeastmaker Yeast Transformation System 2 kit (TaKaRa, Japan). The Ni2+-NTA Superflow resin was purchased from Qiagen (Hilden, Germany). 50× Denhardt solution (B548209-0050) and 20× saline sodium citrate (SSC) (B548109) were purchased from Sangon Biotech (Shanghai, China). Primer synthesis and DNA sequencing were performed by Tsingke (Shanghai, China). Anti-His6 (AbHO012HS01) was purchased from Shanghai HuiOu Biotechnology Co. Ltd (Shanghai, China). Anti-GAPDH (60004-1-Ig) was purchased from Proteintech (Rosemont, IL, USA). A standard ac4C sample (HY-W019670) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).
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