Plasmid Construction and Antibody Validation

Plasmids pcDNA3.1-FLAG-SET8, pcDNA3.1-FLAG-UHRF1, pcDNA3.1-DNMT1, pcDNA3.1-HA-SET8, pcDNA3.1-His-Ub, pcDNA3.1-Myc-LSD1, pcDNA3.1-Myc-USP7 and pPYCAGIP-FLAG-UHRF1 were constructed in our laboratory as previously described (16 (link),38 (link),39 (link)). All mutants were generated by PCR-based point mutagenesis strategy and verified by DNA sequencing. The following antibodies were used in this study: Pan-Kme antibody (Abbkine Abm40195), UHRF1 (proteintech 21402-1-AP), monoclonal DNMT1 (homemade), monoclonal LSD1 (homemade), SET8 (Cell Signaling Technology C18B7), DNMT3A (Santa Cruz sc-20703), p53 (Santa Cruz sc-126), Ub (Santa Cruz sc-8017), GAPDH (Abmart M20006L), β-ACTIN (Sigma A5441), H3 (Abcam ab1791), H3S10P (Epitomics 1173-1), FLAG (Sigma 7425/1804), Myc (Abmart 20002 mouse), and BrdU (Sigma B8434). The following secondary antibody were used: Alexa Fluor 680 goat anti-rabbit IgG (Jackson ImmunoResearch 111-625-144), Alexa Fluor 790 goat anti-mouse IgG (Jackson ImmunoResearch 115-655-146), Alexa Fluor 594 goat anti-rabbit IgG (Jackson ImmunoResearch, 111-585-003) and Alexa Fluor 488 goat anti-mouse IgG (Jackson ImmunoResearch, 115-545-003).

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Zhang H., Gao Q., Tan S., You J., Lyu C., Zhang Y., Han M., Chen Z., Li J., Wang H., Liao L., Qin J., Li J, & Wong J. (2019). SET8 prevents excessive DNA methylation by methylation-mediated degradation of UHRF1 and DNMT1. Nucleic Acids Research, 47(17), 9053-9068.

Publication 2019

DNMT3A GAPDH β-ACTIN FLAG BrdU Alexa Fluor 680 goat anti-rabbit IgG Alexa Fluor 790 goat anti-mouse IgG Alexa Fluor 594 goat anti-rabbit IgG Alexa Fluor 488 goat anti-mouse IgG

Actin Alexa 594 Alexa fluor 488 Anti igg Antibodies Antibody Brdu Dnmt1 Gapdh Goat Lsd1 Mouse Mutagenesis Plasmids Rabbit Usp7

Corresponding Organization : East China Normal University

Other organizations : Soochow University, First Affiliated Hospital of Soochow University, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing Proteome Research Center

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Variable analysis

independent variables

PcDNA3.1-FLAG-SET8
PcDNA3.1-FLAG-UHRF1
PcDNA3.1-DNMT1
PcDNA3.1-HA-SET8
PcDNA3.1-His-Ub
PcDNA3.1-Myc-LSD1
PcDNA3.1-Myc-USP7
PPYCAGIP-FLAG-UHRF1

dependent variables

Pan-Kme
UHRF1
DNMT1
DNMT3A
GAPDH
β-ACTIN
H3S10P

control variables

All mutants were generated by PCR-based point mutagenesis strategy and verified by DNA sequencing.

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