The α4.1 element from the α4 promoter was shown to form at least five distinct complexes involving nuclear proteins. These proteins are expressed in midconfluent RCECs, and their estimated molecular masses range from 39 to 91 kDa. The protein complexes were designated Bp1 to Bp5.
45 The proteins yielding both the Bp4 and Bp5 complexes had molecular masses similar to those reported for both Pax-6 proteins.
33 Because expression of Pax-6 was shown to decrease when RCECs reach a high cell density (see
Fig. 7 ), expression of the Bp1 to Bp5 proteins was examined in nuclear extracts from RCECs cultured at various cell densities. Equal amounts of nuclear proteins from both confluent and midconfluent cells were first SDS-gel fractionated after a protein recovery procedure.
45 Next, proteins from each fraction were tested for their ability to bind the α4.1-labeled probe in an EMSA. Examination of the fractions 3 to 7 was omitted, because their corresponding proteins were unlikely to be Pax-6 proteins. Nuclear proteins from fractions containing Bp2 to Bp5 activities were observed in midconfluent RCECs
(Fig. 8A) . Of the Bp2 to Bp5 proteins, only formation of Bp4 and Bp5 was significantly reduced when RCECs reached postconfluence (compare the results between
Figs. 8A and 8B ). Expression of Bp2 remained unaltered, whereas that of Bp3 was increased when cells reached postconfluence. Only the Bp5 protein, but not any of the other Bp proteins, including Bp4, had a pattern of electrophoretic mobility that matched that of the Pax-6/Pax-6(5a) proteins contained in the chicken embryonic lens extract in EMSA
(Fig. 9A) . To verify whether Bp5 indeed corresponds to Pax-6/Pax-6(5a), EMSAs were conducted in the presence of the Pax-6 antiserum used in
Figure 5B . Nuclear proteins from the fractions shown to support Bp5 binding in both confluent and midconfluent RCECs (fraction 15) were incubated with the α4.1-labeled probe in the presence or absence of 2 μL of the Pax-6 antiserum. The protein fraction 11 that supports Bp2 binding, which has a molecular mass clearly distinct from that of Pax-6 and Pax-6(5a) and whose formation was not significantly altered by cell density, was also used as a negative control. As shown on
Figure 9B , addition of the Pax-6 antiserum did not alter the formation of the Bp2/α4.1 DNA-protein complex from either confluent or midconfluent RCECs. In contrast, formation of the Bp5 complex from midconfluent RCEC nuclear extracts was strongly impaired by the Pax-6 antiserum, whereas no such change was observed using the extract from postconfluent cells. This result is consistent with the lack of any Pax-6 protein detectable by Western blot in the extract from postconfluent RCECs. We conclude that the Bp5 complex formed with the α4.1 probe contains Pax-6 proteins present in midconfluent RCECs.