Figure 1.

Qk5 and Qk6 are the predominant isoforms and can activate splicing in C2C12 muscle cells. (A) Model depicting endogenous Qk protein isoforms (top), with the number of unique amino acids shown at the C terminus (right) and Myc epitope-tagged constructs (bottom) used in subsequent experiments; all Qk proteins share the same 311 amino acids, including Quaking homology 1 (Qua1), Qua2, and hnRNP-K homology (KH) domains. (B) Western blot of whole-cell extracts prepared from undifferentiated C2C12 myoblasts (UD) or C2C12 cells differentiated for 72 h (72h) simultaneously probed with infrared-conjugated secondary antibodies to Qk5 (top), Qk6 (middle), or Qk7 (bottom) in magenta (the notation at the right indicates the migration of specific isoforms) or PanQk in green, which recognizes all Qk isoforms. (C) Indirect immunofluorescence showing Qk isoform-specific (magenta), PanQk (green), and DAPI (blue) staining in C2C12 myoblasts, with overlay of Qk isoform and DAPI at the bottom and a higher magnification inset. The mean signal intensity for each Qk isoform and total Qk (PanQk) is shown at the bottom (±standard deviation), calculated for the total well (n = 9) (see the Materials and Methods for details). Bar, 100 µm. (D) Histogram of log₂ nuclear/cytoplasmic signal intensity of Qk isoforms (X-axis) calculated for each cell imaged in C, with number of cells shown on the Y-axis. (E) Indirect immunofluorescence of C2C12 myoblasts transfected with either Myc-Qk5 (top) or Myc-Qk6 (bottom) stained using anti-Myc epitope antibody (in magenta; left), DAPI (in blue; middle), and both channels overlaid (right). Bar, 20 µm. (F) RT–PCR products from Dup-Capzb exon 9 splicing reporter analyzed on a BioAnalyzer from RNA extracted from C2C12 myoblasts either mock transfected or transfected with Myc-Qk5 or Myc-Qk6 plasmid. The mean values of the percentage included are shown below (±standard deviation) from three independent biological replicates.