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Fig. 1. Control for pre-existing AChR clusters on the
cathode-facing edge of the muscle cell, and an evaluation of
field-induced hotspot reduction. A,A'. The percentage of
untreated cells with hotspots on an edge was recorded. Only
those edge hotspots that resembled FIRPs were counted (see
Materials and methods). The unweighted average (±S.E.M.)
of the percentages obtained from different experiments was
determined, and then halved to account for those that would
have faced the cathode. B,B'. The percentage of 4 Vcm"1
field-treated 1-day-old cells with cathodal clusters. C.C. The
percentage of untreated cells that had hotspots. D.D'. The
percentage of 7-5 Vcm" field-treated 2-day-old cells that had
hotspots. On field-treated cells, AChR staining away from the
cathode-facing edge constituted hotspots. Data base (no.
expts, no. cells): A,C, (3, 162); B, (9, 578); D, (4, 279);
A',C\ (2 ,90); B', (3, 121); D', (3, 109).
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Fig. 2. FIRP formation on a formerly hotspot-free cathodal edge. A. Digitized phase-contrast image showing absence of debris
from cathodal (left) edge. B. Hotspot-free cathodal edge prior to field treatment. C. FIRPs on same edge during 4Vcm~' field
application, 25 min after onset. Note that the AChR staining is elevated in patches, rather than uniformly along the cathode
facing edge of the cell (arrowheads). The bright staining of spherical structures in the middle of the cell and outside the cell is
due to the autofluorescence of yolk platelets. Bar, 10 /im.
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Fig. 3. Control for primary antibody staining at FIRPs. In this example, 1-day-old cells were treated with the 1-h field, 2-h
PFR protocol (see text). A. R-BTX staining showing the FIRP. B. Fluorescein optics showing donkey anti-rabbit staining
following an exposure suitable for rabbit anti-talin staining. Very little nonspecific staining is evident in B. Cathode is towards
the bottom in this micrograph. Bar, 10jUm.
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Fig. 4. 43K protein at hotspots and
FIRPs. A,D,G, R-BTX staining;
B,E,H, the corresponding 43K
staining; C,F,I, the corresponding
phase-contrast views. The top row
shows a muscle cell from an untreated
culture. The staining pattern at the
ventral hotspot (A) matches the pattern
of 43K staining (B) identically.
Following the 1-h field, 2-h PFR (row
2) or the 3-h field, no PFR on 1-day-old
cells (row 3), 43K (E,H arrows) was
elevated at all FIRPs (D,G arrows).
Cathode is to the left in this and all
subsequent micrographs. Bar, 10/tfn.
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Fig. 5. Talin at hotspots and FIRPs. A,D,G, R-BTX staining; B,E,H, the corresponding talin staining; C,F,I, the
corresponding phase-contrast views. The top row shows an example of a typical AChR (A) and talin (B) distribution in a 2-dayold
untreated culture. Talin is always elevated at the ends of the myotomal muscle cells in vitro, independent of hotspot
presence (A,B open arrows). Although there is overlap between the AChR and talin staining patterns (small arrows), there are
regions of AChR staining that do not manifest a similar accumulation of talin (A,B arrowheads). After the 1-h field, 2-h PFR
protocol, talin (E, arrows) was elevated at this FIRP (D, arrows). A particularly robust elevation of talin (H, arrows) was
evident at the FIRP (G, arrows) induced by the 3-h field, no PFR treatment in this 2-day-old cell. A-F, bar, lO^im; G-I,
bar = 6 Jim.
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Fig. 6. 58K protein at hotspots and
FIRPs. A,D,G, R-BTX staining;
B,E,H, the corresponding 58K
staining; C,F,I, the corresponding
phase-contrast views. The top row
shows the colocalization between 58K
(B) and a ventral hotspot (A) in an
untreated 1-day-old cell (C). Unlike
talin, 58K was typically well
colocalized with AChR domains in
hotspots. S8K proteins (E,H arrows)
was elevated at virtually all FIRPs
(D,G arrows) induced by both the 1 h
field, 2-h PFR protocol (row 2) and
the 3-h field, no PFR protocol in
1-day-old cells (row 3). Bar, 10 fun.
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