Figure 1

PTP inhibition by pervanadate reduces the stability of agrin-induced AChR clusters in C2C12 myotubes. (A) Myotubes were treated with agrin (1 nM) for 6–8 h, after which PTPs were blocked with 20 μM pervanadate at the point of agrin withdrawal (Agrin + Withdr. + PV) for a withdrawal period of 16 h. In controls, cultures were left untreated (No Agrin), treated with agrin alone for 6–8 h (Agrin), or treated with agrin followed by withdrawal for 16 h (Agrin + Withdr.). Cells were also treated with agrin for 6–8 h + 16 h, and this produced the same amount of clusters as a 6–8 h agrin incubation (data not shown). AChRs were stained with rhodamine-α-BTX and analyzed by fluorescence microscopy. Scale bar, 40 μm. (B) The number of AChR clusters per field was calculated using ImageJ software, using fixed intensity thresholds (150–255) and minimum area of 100 pixels occupied by a cluster. The number of AChR clusters per field (400× magnification) is shown as the percentage of clusters in agrin-treated cells (Agrin) (means ± SEM, N = 50 from four similar experiments). Phosphatase inhibition significantly decreases the number of pre-existing, agrin-induced AChR clusters following agrin removal (** p < 0.01; two-tailed paired t test).