Figure 2. SCRIB spectrin binding motifs bind to CH1 domains of the β spectrin family.
(A) Schematic representation of full length human β2 spectrin protein (left) and different β2 spectrin deletion constructs used in the study (right). (B) GST pull down using a GST-SCRIB C1 resin from HEK293 cell lysates expressing indicated GFP-tagged β2 spectrin CH domains. Samples were analyzed by ponceau staining and immunoblotting using anti GFP. (C) Fluorescence images showing HeLa cells expressing GFP-SCRIB C1 construct together with RFP-NCH1CH2 or SID β2 spectrin domains and stained with phalloidin. (D) GST-SCCRIB C1 pull down from HEK293 cell lysates expressing indicated GFP tagged β2 spectrin CH domains. Samples were analyzed by ponceau staining and immunoblotting using anti GFP. (E) Phylogenetic analysis of the Calponin Homology (CH) domains type1, 2 and 3 used in this study. (F) GST pull down from HEK293 cell lysates expressing the indicated CH domains using a GST-SCRIB SADH3 resin. Pull down was done in low or high stringency conditions (LSB: Low Salt Buffer, HSB: High Salt Buffer). Ponceau staining indicates the relative amount of GST tagged proteins bound to the resin. Samples were analyzed by ponceau staining and immunoblotting using anti GFP. Bars, 10 µm.