Splicing factor proline- and glutamine-rich (SFPQ) is an essential RNA-binding protein (RBP) with diverse nuclear functions. In neurons, tight regulation of SFPQ is particularly important, as altered SFPQ function or mislocalization has been implicated in several neurodegenerative diseases. These alterations are often linked to changes in gene expression or in the phosphorylation state of SFPQ. TIMAP (TGF-beta-inhibited membrane-associated protein) is a regulatory subunit of protein phosphatase 1 (PP1). Our earlier work identified SFPQ as a nuclear interacting partner of TIMAP. Pathway enrichment analysis of transcriptomic changes following TIMAP depletion in SH-SY5Y cells revealed significant enrichment of processes associated with neuronal morphogenesis, projection development, and neuronal differentiation, all of which are biological functions in which SFPQ has well-established functions. SFPQ has reported phosphorylation sites at S8, S283, and T687, suggesting it may be regulated by the TIMAP-PP1c phosphatase complex. Our aim was to investigate whether the TIMAP-PP1c phosphatase complex regulates SFPQ through phosphorylation. Interaction between SFPQ and the TIMAP-PP1c complex was verified by pull-down assay, using recombinant PP1c and TIMAP proteins, and additionally, by immunoprecipitation of SFPQ from SH-SY5Y. Next, we have cloned the SFPQ coding sequence into pCMV-HA mammalian expression vector, and using site directed mutagenesis, T687A phosphonull and T687D phosphomimic mutants of SFPQ were created. TIMAP depleted (shTIMAP), and pLKO control (shCTRL) SH-SY5Y cells were transfected with wild-type and phosphomutant SFPQ encoding plasmids using Lipofectamine 3000 reagent. Western blot analysis in both cell lines confirmed the overexpression of all the recombinant proteins. Since the phosphorylation state of SFPQ can influence its subcellular localization, we performed immunofluorescent staining on transfected shCTRL and shTIMAP cells. Confocal microscopy revealed differences in the subcellular distribution of the SFPQ mutants, which will be further evaluated by cell fractionation. In conclusion, we validated that the TIMAP-PP1c phosphatase complex interacts with SFPQ and successfully created wild-type and T687 phosphomutants SFPQ clones, which can be used to explore the potential role of this complex in regulating SFPQ.