Figure 1. Differential overexpression of PTB, nPTB and ROD1.

A) Expression constructs featured a CMV promoter, common 5′ and 3′ UTRs and an N-terminal Xpress epitope tag. Open reading frames (ORF) for PTB and its paralogs were the only variable between constructs. nPTB* is the codon-optimized nPTB expression construct described in the text. B) Western blots of 293 cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. Positions of size markers (kDa) are indicated to the right. C) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected 293 cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). D) Nuclease protection assay of cytoplasmic RNA harvested from 293 cells transfected with the constructs indicated above. 4Z is a mock transfection (pGEM4Z). “T” is the test probe, corresponding to the Xpress probe region of the expression construct, while “C” is a control probe detecting the cotransfected GFP construct. Positions of size markers (nt) are indicated to the left. E) Western blots of L cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. 4Z is a mock transfection (pGEM4Z). F) Nuclease protection of cytoplasmic RNA from L cells. Details as in “D”. G) Western blots of HeLa cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. H) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected HeLa cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). I) Nuclease protection of cytoplasmic RNA from HeLa cells. Details as in “D”.