Journal: BMC Plant Biology
Article Title: SUPERMAN requires its EAR motif to recruit the corepressor TOPLESS and to function as a cadastral regulator in floral patterning
doi: 10.1186/s12870-025-07553-z
Figure Lengend Snippet: CRISPR-generated EAR mutations disrupt physical interaction between SUP and TPL. ( A ) Yeast two-hybrid assays testing interaction between TPL [fused to the GAL4 DNA-binding domain (DBD)] and wild-type SUP (DLELRL), mSUP (DLELL) or mSUP (∆EAR) [each fused to the GAL4 activation domain (AD)]. Growth on selection (right panel) indicates physical interaction between TPL and wild-type SUP (DLELRL) only. ( B ) In vitro pull-down assays using GST-tagged N-TPL show a strong interaction with SBP-tagged wild-type SUP (DLELRL) which is weakened by mutation of the EAR motif in CRISPR allele mSUP (DLELL). Ponceau S staining shows efficient GST protein expression. Full-length blots and quantification of Western blot signal intensities are presented in Supplementary Fig. S7
Article Snippet: To verify yeast protein expression, Western blots were stained with 0.1% Ponceau S in 5% v/v acetic acid, washed in water, and analyzed using anti-GAL4 DBD (sc-577) or anti-GAL4 AD (sc-1663) antibodies (Santa Cruz Biotechnologies, Dallas, TX, USA).
Techniques: CRISPR, Generated, Binding Assay, Activation Assay, Selection, In Vitro, Mutagenesis, Staining, Expressing, Western Blot