Calf intestinal alkaline phosphatase

The global DNA methylation levels were determined by measuring the 5-methyl-cytosine level with a Global DNA Methylation competitive enzyme-linked immunosorbent assay (ELISA) kit from Cell Biolabs (San Diego, CA, USA). Briefly, genomic DNA was extracted from cell samples using the Wizard® Genomic DNA purification kit (Promega, Madison, WI, USA). A total of 1 μg genomic DNA (0.1 μg/μL concentration) was denatured into single-stranded DNA by heating the samples at 95° C for 10 mins, followed by quick cooling on ice. Next, 1 μL of nuclease P1 (New England Biolabs, Ipswich, MA, USA) and an appropriate amount of nuclease P1 reaction buffer (New England Biolabs, Ipswich, MA, USA) were added to single-stranded DNA samples, and the whole mixture was incubated at 95° C for 1 hr to digest DNA into nucleosides. After that, the samples were treated with calf intestinal alkaline phosphatase (Takara Bio Inc., Shiga, Japan) for 1 hr at 37° C. Further steps were conducted according to the manufacturer’s recommendation. Sample absorbance was measured at 450 nm using a microplate reader (DTX880, Beckman Coulter, Inc., California, USA).

PrimeSTAR polymerase, restriction enzymes, calf intestinal alkaline phosphatase, Dpn I, T4 DNA ligase, In-Fusion HD Cloning Plus kit and vector (pMD18-T) were purchased from Takara (Dalian, China). The plasmid mini-prep kit, PCR purification kit, and the agarose gel DNA purification kit were purchased from Tiangen Co. Ltd (Beijing, China). DNA sequencing and DNA primer synthesis were performed by Shanghai RuiDi Biological Technology Co. Ltd. (Shanghai, China). Tryptone and yeast extract were obtained from Oxoid (Hampshire, UK). β-cyclodextrin was purchased from Sigma-Aldrich (Milwaukee, WI, USA). Non-fat powdered milk was purchased from BBI Life Sciences (Shanghai, China). Other chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

Gel shift assay was performed as previously described with minor modifications [25 (link)]. HEK 293T cells were grown in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Life Technologies, Grand Island, NY) containing 10% fetal bovine serum in 3.5 cm plates and transfected with Arx expression vectors using polyethyleneimine (PEI) (Polysciences). After a brief sonication in 150 μl of phosphatase buffer (50 mM Tris [pH 8.0], 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.1% sodium dodecyl sulfate [SDS], 10% glycerol, 1 mM dithiothreitol [DTT], 0.1 mM phenylmethylsulfonyl fluoride [PMSF]), the cells were centrifuged at 12000 rpm for 10 min. Supernatants (50 μl) were incubated with 10 U of calf intestinal alkaline phosphatase (CIAP) (Takara) for 30 min at 30°C. As a control, the same reaction was performed in the presence of 60 mM Na₂HPO₄, which competed for the phosphatase reaction. The total protein concentration of the supernatants was determined by the BCA Protein Assay Kit (Pierce); equal amounts of protein were used for the gel shift assay. ARX protein expression was detected by the relevant tag antibody. The gel shift assay for the treatment with Calyculin A (CA, Cell Signaling Technology) was similar as the CIAP treatment experiment: after 24 h of the transfection, cells were treated with 100 nM CA for 30 min before lysis, a DMSO (Sigma–Aldrich) treated group was used as the control.