Figure 8
Gel-shift analyses with 32P-HSEαB and its mutants. Post-natal day 10 rat lens nuclear extracts, which predominantly contain HSF4 [25], were used for these assays. Autoradiograph shows the complex III (lane HSEαB), the major complex, which contains trimeric HSF4, known to be associated with the active promoter. The two minor complexes (I and II), seen mostly when the heat shock promoter is not active, are not seen here clearly. TSS transcription start site. M1–M3 are probes with mutations in one or two of the 5′-NGAAN-3′ motifs (arrows) of the trimeric HSE (blue), as expected [24, 25] they do not bind HSF4. M4 and M4X are two HSEαB mutant probes with alterations in the GPS. Both these probes have diminished HSF4 binding activity (complex III) when compared to wild-type probe, HSE-αB. All mutations are shown in red and are underlined. All the probes are 30-bp long and had comparable specific activities.