Figure 7.

Confirmation of the binding of SF9 to an epitope within the common SID. A. Schematic alignment of the full-length human non-muscle myosin IIA heavy chain (NMHCAIIA) protein, of the three found SID and of the constructs made to confirm SF9 binding. The common (29aa long) SID is depicted in red. B. Analysis using immunofluorescence. HeLa cells were transfected with full-length NMHCAIIA (a-c) or the NMHCIIA-ΔC-ter fragment expressing only the first 1338 amino acids and lacking the common SID (d-f). Only the full length but not the truncated recombinant GFP-tagged protein was recognized by hSF9 (yellow in panel c versus green in panel f). Bar 10 μm. C. Analysis using immunoblot. HeLa cells were transfected with either a GFP-tagged NMHCIIA fragment containing the common SID (fragment, a), or a GFP-tagged NMHCIIA fragment deleted from the common SID (ΔSF9BD, b). Immunoblot analysis of total cell extracts showed that hSF9 only detected the larger fragment (lane a) while the anti-GFP antibody recognized both recombinant proteins at ~70 kDa. D. Sequence alignment of the 29-amino-acid long human non-muscle myosin IIA SID with the corresponding insect protein. Note the high level of similarity. E. Specific binding of SF9 to the drosophila protein. Total cell extract of drosophila (S2) cells incubated (+) or not (-) with RNAi to inactivate the expression of zipper (the unique myosin II homologue in drosophila) were analyzed by immunoblot. A specific band at ~200 kDa is present only in non treated cells. Tubulin stained by an anti α-tubulin antibody was used as loading control.