Immunofluorescence Staining of 6mA and HA

Immunofluorescence (IF) staining experiments followed previously described procedures (19 (link),42–44 (link)). For the single antibody staining, the primary antibodies were α -6mA (Synaptic Systems, 202003, 1:2000) (19 (link)) and α -HA (Cell Signaling, #3724, 1:200) and the secondary antibody was Goat anti-Rabbit IgG (H+L) (Invitrogen, A-21428, 1:4000). For the co-staining, cells were first incubated with α -HA (Covance, MMS-101P, 1:500, mouse) and its secondary antibody (goat anti-mouse IgG (H+L), Alexa Fluor 555, Invitrogen, A-21127, 1:4000). After crosslink by 3% PFA, α -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (Goat anti-Rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used. Digital images were collected using an Olympus BX43 microscope with an Olympus DP72 camera.

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Wang Y., Sheng Y., Liu Y., Zhang W., Cheng T., Duan L., Pan B., Qiao Y., Liu Y, & Gao S. (2019). A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription. Nucleic Acids Research, 47(22), 11771-11789.

Publication 2019

α -6mA Goat anti-Rabbit IgG (H+L) MMS-101P goat anti-mouse IgG (H+L), Alexa Fluor 555 goat anti-rabbit IgG (H+L), Alexa Fluor 488 BX43 microscope DP72 camera

Alexa fluor 488 Alexa fluor 555 Anti igg Antibodies Antibody Cells Goat Immunofluorescence Microscope Mouse Rabbit

Corresponding Organization : Ocean University of China

Other organizations : Anhui Agricultural University, University of Michigan–Ann Arbor

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Variable analysis

independent variables

Primary antibody α-6mA (Synaptic Systems, 202003, 1:2000)
Primary antibody α-HA (Cell Signaling, #3724, 1:200)
Primary antibody α-HA (Covance, MMS-101P, 1:500, mouse)

dependent variables

Immunofluorescence staining patterns

control variables

Experimental procedures followed previously described protocols (19, 42-44)
Secondary antibodies: Goat anti-Rabbit IgG (H+L) (Invitrogen, A-21428, 1:4000), Goat anti-mouse IgG (H+L), Alexa Fluor 555 (Invitrogen, A-21127, 1:4000), Goat anti-rabbit IgG (H+L), Alexa Fluor 488 (Invitrogen, A-11008, 1:4000)
Fixation with 3% PFA

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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