Fig. 2. AltSFPQ encodes a previously unidentified SFPQ protein.

(A) Polysome profiling schema, with example absorbance profile. RNP, ribonucleoprotein. (B) Analysis of SFPQ transcripts and GAPDH mRNA by qPCR from hiPSC-derived NPCs (DIV7), plotted as the percentage of total (n = 3; one cell line derived from a healthy donor and two cell lines derived from a patient with VCP-ALS). (C) Polysome profiles obtained from hiPSC-derived DIV14 NPCs with (red) or without (black) EDTA treatment; EDTA treatment caused substantial loss of polysomes. Analysis (right) of wtSFPQ or altSFPQ mRNA expression by qPCR from each fraction, plotted as the percentage of total (n = 3; two cell lines from healthy donors and one cell line from a patient with VCP-ALS). (D) Schematic depiction of SFPQ isoforms and antibodies used for IP and immunoblotting. One antibody recognizes an epitope common to both proteins (“N”-term SFPQ antibody), whereas a C-terminally targeting antibody recognizes only wtSFPQ (“C”-term SFPQ antibody). RRMs, RNA recognition motifs. (E) Western blot showing the altSFPQ isoform expression in SMG1i-treated HEK293T cells; n = 3, stars demarcate the proteins. (F) AltSFPQ mRNA levels (qPCR, normalized over GAPDH) in nontargeting control ASO (NTC)–treated and ASO20-treated HEK293T cells (n = 3; unpaired t test). (G) Representative Western blot of the endogenous SFPQ protein expression in NTC-treated or ASO20-treated HEK293T cells; relates to (F). Initial IP using ab177149 (C terminus targeting) SFPQ antibody removes wtSFPQ and enables visualization of the altSFPQ protein in “cleared” fractions (using N-terminal SFPQ antibody). An 8-μg input was loaded along with increasing amounts of cleared lysate; an input equivalent amount of the bead eluate was loaded. (H) Quantification of altSFPQ (normalized over vinculin) in (G), using the highest-quantity cleared lysate within each replicate (n = 3; unpaired t test). Graphs are presented as means ± SEM.