Fig. 3. AltSFPQ encodes a cytoplasm-predominant protein, which attenuates subcellular distribution of DBHS proteins.

(A) Representative immunofluorescence images showing the subcellular localization of HA-tagged recombinant SFPQ proteins in transfected HEK293T cells. (B) Western blot of nuclear-cytoplasmic fractionated SMG1i-treated HEK293T cells; vinculin and lamin B1 act as subcellular markers. (C) Quantification of the nuclear-cytoplasmic ratio of SFPQ proteins in fractionated SMG1i Western blots; the C-term SFPQ antibody signal is used to quantify wtSFPQ, and the N-term antibody is used to quantify the smaller visible SFPQ protein (i.e., altSFPQ); n = 3; one-way ANOVA, Tukey’s multiple comparisons. (D) HEK293T cells were transfected with 100 ng of eGFP-wtSFPQ (+100 ng of EV plasmid), 100 ng of mAPPLE-wtSFPQ (+100 ng of EV plasmid), or 100 ng of each tagged SFPQ protein; representative zoomed-in images are displayed (arrowheads show co-localized cytoplasmic eGFP and mAPPLE signals; stars show the nuclei exhibiting both eGFP and mAPPLE signal). (E) Image analysis quantification relating to (D), measuring the nuclear-cytoplasmic ratio (using a 20-μm perinuclear ring region) of the eGFP signal (reflecting the eGFP-wtSFPQ protein) in eGFP-wtSFPQ transfected versus eGFP-wtSFPQ/mAPPLE-altSFPQ cotransfected cells (n = 3; each data point represents the average across ≥5 fields of view for each replicate; unpaired t test). a.u., arbitrary units. (F) As for (E) but assessing the nuclear-cytoplasmic ratio of the mAPPLE signal (reflecting the mAPPLE-altSFPQ protein) (n = 3; each data point represents the average of ≥5 fields of view for each replicate; unpaired t test).