Fig. 3.
Expression of MAPK at the NMJ. A, Western blot of body wall muscle extracts stained with anti-DmERK-A (left) and reprobed with anti-DpMAPK (middle) after stripping. To show that the blot was completely stripped after staining with DmERK-A, the second lane of the middle panel was incubated with secondary antibody and chemiluminescent reagent before exposure to film. Each lane in the right andmiddle panels was loaded with an equal amount of protein from body wall muscle extracts (∼5 body wall muscle preparations perlane). In the Western blot shown in the left panel, equal amounts of protein (∼2.5 body wall muscle preparations per lane) from extracts of wild-type andrl10A/+ heterozygotes were loaded, and the membrane was probed with anti-DmERK-A.B, Western blots of body wall muscle extracts from wild type (WT) and from larvae overexpressing Ras1V12S35 (V12S35), wild-type Ras1 (Rwt), Ras1V12G37 (V12G37), and Ras1V12C40 (V12C40) using C380, probed sequentially with anti-DpMAPK and anti-DmERK-A. Equal amounts of protein were loaded in each lane (3.5 body wall muscle preparations perlane). The molecular masses given at theright of the blots are in kilodaltons. C, Anti-DmERK-A immunoreactivity at type I synaptic boutons.D, Antibodies against activated MAPK (DpMAPK) result in highly immunoreactive hot spots at synaptic boutons. E, DpMAPK hot spots colocalize with DmERK-A patches at the boutons, but DpMAPK staining is more restricted than DmERK-A, as shown by mergingA and B. Scale bar, 10 μm.