Fig. 1. Expression of 4 constructs. (A) A4EX recognizes the extracellular domain of Xenopus integrin 4. Embryos injected with full-length Xenopus 4 (4wt) or partial cytoplasmic tail deletion 4 mutant (4RR) were lysed and subjected to western blot with A4EX (raised against the 4 extracellular domain) and D2AP (raised against the 4 cytoplasmic tail). Protein equivalent to one embryo was loaded per lane. Arrows indicated bands of 140 kDa, 80 kDa and 60 kDa. A4EX can detect both 4wt and 4RR, whereas D2AP only recognizes 4wt. (B) 4 constructs are expressed on embryonic cell surface. Cell-surface biotinylated 4 tail truncation and chimera proteins (as indicated) are immunoprecipitated by A4EX. Each lane contains protein precipitated from seven embryos. Arrows indicate bands of 80 kDa and 60 kDa. Note that the 4 cytoplasmic tail deletion mutants and chimeras are present at the cell surface predominantly in the cleaved form. 42 is expressed on the surface less well than other constructs. Quantification of the pixel densities of the 4 bands indicates a less-than-twofold variation in surface expression from sample to sample, with the exception of 42. (C) 4 constructs form heterodimers with the ß1 subunit. Embryos co-injected with RNA transcripts of 4 constructs and ß1 were lysed at stage 15 and immunoprecipitated with mAb 8c8. The immunoprecipitated proteins were then separated on an 8% polyacrylamide gel and western blotted with A4EX. Precipitate equivalent to ten embryos was loaded per lane. Both full-length and cleaved forms of 4 can associate with ß1 subunit.

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