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. 2007 Jul 2;8:46. doi: 10.1186/1471-2202-8-46

Figure 2.

Figure 2

AChR cluster stability is rescued by PTP inhibition in src-/-;fyn-/- myotubes and PP2-treated C2C12 myotubes. (A) PTPs were blocked with 20 μM pervanadate in src-/-;fyn-/- myotube cultures at the point of agrin withdrawal for a period of 5 h (Agrin + Withdr. + PV), following agrin treatment for 16 h. As controls, myotubes were either left untreated (No Agrin), treated with agrin for 16 h (Agrin) or treated with agrin followed by withdrawal for 5 h (Agrin + Withdr.). AChRs were stained with rhodamine-α-BTX and analyzed by fluorescence microscopy. Scale bar, 40 μm. White arrowheads indicate AChR clusters. (B) The number of AChR clusters per myotube is shown as the percentage of clusters in agrin-treated src-/-;fyn-/- cells (Agrin) (means ± SEM; N = 15 from three similar experiments; **p < 0.01, ***p < 0.001 by two-tailed paired t test). Pervanadate restores cluster stability following agrin withdrawal in src-/-;fyn-/- myotubes. (C) The inhibitor PP2 was used to block SFK activity in C2C12 myotubes. Myotubes were treated with agrin for 6–8 h in the presence of 10 μM PP2 for the last 2 h (these 2 h were chosen to ensure that PP2 was effective at the point of agrin withdrawal). Agrin was removed and myotubes incubated with agrin-free medium in the presence of pervanadate and PP2 for 16 h (Agrin + Withdr. + PP2 + PV). In controls, cultures were treated with agrin followed by withdrawal for 16 h in the presence or absence of PP2 (PP2 was added 2 h before agrin removal) (Agrin + Withdr.; Agrin + Withdr. + PP2). AChRs were stained with rhodamine-α-BTX, analyzed by fluorescence microscopy. Scale bar, 40 μm. (D) The number of AChR clusters per field (400× magnification) was calculated, as described in Figure 1, as the percentage of agrin-treated C2C12 cells (Agrin) (not shown) (means ± SEM, N = 30 from three experiments). *** indicates significant difference to the other two bars (p < 0.001; two-tailed paired t test). Blocking SFKs with PP2 reduces the stability of pre-existing clusters in C2C12 myotubes, and pervanadate restores the stability to normal levels.