Figure 2.

PRTG is expressed in early committed cells, and blockage of PRTG activity promotes neuronal differentiation in P19 cells. A, B, Differentiation of P19 cells was first induced by retinoic acid for 4 d (Day 1–4), and then the cells were cultured in serum-free medium for another 4 d (Day 5–8). A, Expression of prtg mRNA was analyzed by the Northern blot. MAP2, A neuronal marker; cyclophilin, RNA loading control. B, Expression of PRTG, Oct4, Sox1, and MAP2 in differentiating P19 cells was analyzed by Western blot. A nonspecific band with molecular weight 40 kDa recognized by α-PRTG2 was used as protein loading control (Ctrl). C, Quantification of protein amounts from the Western blots shown in B. Relative protein expression levels are normalized to the highest protein expression amount of each protein. D, Lysates of HEK293T cells transfected with shPRTG(m), control vector (UI4), or shS together with pmPRTGf were subjected to Western blot (WB) by anti-myc and anti-β-tubulin antibodies. E, Lysates of HEK293T cells transfected with pmPRTGf(myc), prPRTGf(HA), and prPRTGΔc(HA) were immunoprecipitated by anti-HA or anti-myc antiserum and then subjected to Western blot by anti-myc or anti-HA antiserum. F, Experimental procedure for P19 cell neuronal differentiation analysis. P19 cells were transfected with 0.4 μg of pAscl1 and 1.6 μg of the indicated plasmids, subjected to selection with G418 or puromycin, and then cultured in SF21 serum-free medium for 4 d. G, Differentiated neuronal cells were detected by TuJ1 staining. Scale bar, 50 μm. H, The percentage of cells labeled by TuJ1 in one whole well of the six-well plate was counted. Data are then normalized relative to the percentage of the control (pEF1A or UI4) and shown as the mean ± SEM (n = 3; **p < 0.01, by Student's t test).