Fig 2: Subcellular localisation of ZFP804A and NT5C2 in cortical neurons. (a–c) Western blotting of cell fractions generated from mouse cortex. S1, extranuclear cell lysate; S2, cytosol; P2, crude membrane/synaptic fraction; P2S, membrane/synaptic supernatant, P2P, membrane/synaptic precipitate. (a) Immunoblotting of cortical cell fractionations for PSD‐95 and ZFP804A with β‐actin loading control. PSD‐95 was enriched in the P2 fractions, while ZFP804A was present in all fractions but enriched in S2 cytosolic fraction. (b) Immunoblotting of detergent treated crude synaptic fractions of mouse cortex, for ZFP804A, PSD‐95 and SV2A with a β‐actin loading control. PSD‐95 was enriched in the Tx P2P and RIPA P2P fractions, while SV2A and ZFP804A were predominately present in Tx P2S and RIPA P2S soluble fractions. (c) Immunoblotting of cortical cell fractionations for ZFP804A and NT5C2 with a β‐actin loading control. ZFP804A and NT5C2 were present in all fractions; ZFP804A and NT5C2 were abundant in S2 fractions compared to P2. ZFP804A and NT5C2 were present more in TritonX‐100 soluble fractions (Tx P2S) to TritonX‐100 insoluble fractions (Tx P2P). PSD‐95 was used to demonstrate synaptic enrichment. (d) Representative confocal image of a section of dendrite from DIV20 cortical neurons transfected with eGFP constructs and co‐stained for PSD‐95, ZFP804A and NT5C2. Yellow arrows indicate the localisation co‐localisation of ZFP804A and NT5C2 with PSD‐95 along the synapses. Further confirmation was also conducted with orthogonal analysis. (e) Representative confocal image of a section of dendrite from DIV20 cortical neurons immunostained for MAP 2 (morphological marker) and a previously validated antibody for ZFP804A. Quantification of ZFP804A puncta across 10 μm2 in dendrites or crude synaptic region is shown in a box and whisker plot, showing maximum and minimum values. (f) Representative confocal image of a section of dendrite from DIV20 cortical neurons immunostained for MAP 2 (morphological marker) and NT5C2. Quantification of NT5C2 puncta across 10 μm2 in dendrites or crude synaptic region is shown in a box and whisker plot, showing maximum and minimum values. n = 51 cells from three independent experiments.

Fig 3: NT5C2 and ZFP804A co‐localise and form a protein complex near synapses in rat cortical neurons. (a) Representative confocal image of DIV 20 cortical neurons immunostained for MAP 2 (morphological marker), NT5C2 and ZFP804A. Orange arrows indicate the co‐localisation of NT5C2 and ZFP804A along dendrites, whereas white arrowheads indicate co‐localised puncta in synaptic regions. XZ and YZ orthogonal view further demonstrate co‐localisation of both proteins (orange arrows). (b) Pearson's coefficient (r) quantification for the extent of co‐localisation between ZFP804A and NT5C2 in DIV 20 primary cortical neurons. Co‐localisation of ZFP804A and NT5C2 was confirmed since r was >0, thus showing a positive correlation between ZFP804A and NT5C2 puncta intensity overlap. Values >0 suggest positive co‐localisation for both puncta, with significantly more ZFP804A in NT5C2 (parametric unpaired t‐test ****p < 0.0001), indicating a positive correlation between the overlapping intensities of ZFP804A and NT5C2 puncta across dendritic and synaptic regions; n = 52 cells, three biological replicates. (c) Quantification of relative intensities of ZFP804A in NT5C2 versus NT5C2 in ZFP804A across dendritic and crude synaptic regions using Mander's coefficient. Significantly more ZFP804A puncta were found in NT5C2 (parametric unpaired t‐test ****p < 0.0001, n = 51 cells, three biological replicates). (d and e) Quantification of co‐localisation: (d) density of NT5C2 puncta across an area of 10 μm2 positive for ZFP804A and (e) density of ZFP804A puncta positive for NT5C2 staining along dendrites or within synaptic regions. n = 51 cells, three biological replicates. (f) Co‐immunoprecipitation (co‐IP) assay of DIV 20 cortical neurons. Cell lysates were immunoprecipitated with either an antibody against ZNF804A/ZFP804A or against rabbit IgG (control). Samples were subjected to immunoblotting and then subsequently probed with antibodies for NT5C2 and ZNF804A. A specific band for ZFP804A was detected in the input, ZNF804A‐IP and flow through lanes as expected—no band was detected in the rabbit IgG‐IP lane, indicating the specificity of the assay. Probing with an antibody against NT5C2 revealed bands in the input, ZNF804A IP and flow through lanes, but not the rabbit IgG‐IP lane. This indicates that NT5C2 is part of a protein complex with ZNF804A.

Fig 4: Knockdown of Zfp804a causes the redistribution of NT5C2. (a) Representative confocal image of DIV20 cortical neurons either untransfected (blank) or transfected with a scramble siRNA (scramble) or an siRNA for Zfp804a (Zfp804a siRNA). Cells were immunostained for MAP 2 (morphological marker), an antibody against ZFP804A (green) or NT5C2 (green). (b) Quantification of ZFP804A linear density in blank, scramble or Zfp804a siRNA conditions. ZFP804A linear density is shown as a box and whisker plot, with minimum and maximum values. n = 15–18 cells from four independent experiments. (c) Quantification of NT5C2 linear density along dendrites or crude synaptic regions, in blank, scramble or Zfp804a siRNA conditions. The box and whisker plot show minimum and maximum. n = 18 cells from four independent experiments.

Fig 5: NT5C2 and ZNF804A form a protein complex and co‐localise when expressed in HEK293 cells. (a) Representative confocal image of HEK293T cells expressing GFP‐ZNF804A and co‐stained for F‐actin. Ectopically expressed GFP‐ZNF804A localises to cell cytoplasm and plasma membrane (yellow arrows). Near the plasma membrane, GFP‐ZNF804A co‐localises with F‐actin. (b) Representative confocal image of HEK293T cells expressing Myc‐NT5C2 and co‐stained for F‐actin. Ectopically expressed Myc‐NT5C2 localises to cell cytoplasm (yellow arrows) like previous descriptions. (c) Confocal images of untransfected HEK293T cells, or cells co‐transfected with GFP‐ZNF804A and Myc‐NT5C2. In co‐expressing cells, GFP‐ZNF804A and Myc‐NT5C2 co‐localise to the cell cytoplasm and near the plasma membrane (yellow arrows). Yellow dotted box indicates area magnified in (d). (d) (i) + (ii) Intensity plot and magnified image of dotted box from (c), demonstrating co‐localisation of GFP‐ZNF804A and Myc‐NT5C2. (e) Co‐immunoprecipitation (co‐IP) assay of HEK293T cells co‐expressing GFP‐ZNF804A and Myc‐NT5C2. Cell lysates were immunoprecipitated with a Myc antibody to isolate NT5C2 and the interacting partner. Samples were subjected to immunoblotting and then subsequently probed with antibodies for Myc (to detect Myc‐NT5C2) and GFP (to detect GFP‐ZNF804A). In the lower blot, ‘#’ denotes non‐specific IgG bands, and the red ‘*’ indicates Myc‐NT5C2. A specific band for GFP‐ZNF804A was detected in immunoprecipitated cell lysates expressing both proteins but not from untransfected cell lysate. (f) Co‐IP assay of HEK293T cells co‐expressing GFP‐ZNF804A and Myc‐NT5C2, whereby cell lysates were incubated with control normal rabbit IgG. Scale bar = 50 μm for (a) and (b) and 10 μm for (d).