Skip to main content
. 2000 Jul;11(7):2335–2347. doi: 10.1091/mbc.11.7.2335

Figure 3.

Figure 3

Purified 6His-Skn7p can specifically bind HSEs in vitro. EMSA was performed with the use of E. coli–expressed affinity-purified 6His-Skn7 protein and a probe comprising a double-stranded oligonucleotide corresponding to the 26-base pair HSE2 region of the SSA1 gene. The specificity of binding was assessed by the addition of cold HSE2 probe (HSE: tcgaTTTTCCAGAACGTTCCATCGGC) at 5-, 10-, and 50-fold molar excess, compared with the addition of a mutated HSE (MUT HSE: tcgaTTTTCCAAAACGTTTCATCGGC) at 10-, 50-, and 100-fold molar excess. Mutation of the underlined nucleotides in the consensus HSE abolishes Hsf1 binding to the sequence. Polyclonal anti-Skn7 antibody (α-Skn7) at a 1:100 dilution or preimmune serum at the same concentration (Pre Imm) was added to the binding reaction 15 min before the addition of labeled probe. Free probe without the addition of protein migrated off the gel and is indicated (Free).