Figure 5
Comparative EMSA of nuclear extracts with [32P]-Rab16A promoter (300 bp natural promoter, containing motif I, motif IIa and IIb) as probe. A. Formation of two different complexes, C1 and CII (arrow marked) differing in their mobility was observed when equal amount of nuclear extract (20 μg) from M-1-48 and Nonabokra were incubated with equal amount of [32P]-labeled Rab16A promoter (80,000 CPM) as probe. Complex formation was extremely low and inducible in M-1-48 (lane 2 and 3) whereas both complexes were high and constitutive in Nonabokra (lanes 4 and 5). B. Specificity of the complexes formed in EMSA, as shown by the competition by non-labeled Rab16A at 100 fold molar excess for CI complex (lane 3) and at 250 fold molar excess for CII complex. 100 fold excess of 1XABRE (lane 4) showed competition with CI complex formation, suggesting motif I is equivalent to ABRE. C. Nuclear extract (20 μg) from Nonabokra control lamina showed the formation of CI and CII with [32P]Rab16A promoter (lane 2). The figure shows that both pre- and post-incubation (lane 4 and 5) with antiserum (before and after the addition of [32P]-Rab16A promoter) abolished CI, the faster migrating complex. Incubation with equal concentration of pre-immune serum shows no such effect (Lane 3). D. EMSA of [32P]Rab16A promoter with the 43 kD recombinant 6X His-OSBZ8FL protein shows the formation of a single complex and the shift was equal to CI formed by the nuclear extract and [32P]Rab16A (Lane 3). Lane 2 shows the usual formation of CI and CII with Nonabokra laminar nuclear extract.