Epitope mapping and nanobody functionality as intrabody. (A) Schematic representation of GST-fusion proteins used in pull-down experiments. (B) Epitope mapping. Glutathione-S-transferase pull-down assays with recombinant CAPNbs (left, input) and GST-CapG fragments (indicated on top) were evaluated with SDS-PAGE and coomassie staining. (C) Co-immunoprecipitation. EGFP-tagged CAPNbs or a GFP control (numbers indicated on top) were stably expressed in MDA-MB-231 cells and detected in cell extracts (top panel). They were retrieved from cell extracts with a polyclonal anti-EGFP antibody (bottom panel). All CAPNbs immunoprecipitated endogenous CapG (middle panel). CE, crude extract. (D) Upper panels: MDA-MB-231 cells were transfected with EGFP (control) and stained for endogenous CapG (red) and show the typical nucleocytoplasmic distribution of CapG. Middle panels: MDA-MB-231 cells were transfected with CAPNb2-EGFP coupled to the SV40 large T-antigen nuclear-localization sequence (NLS), promoting a stronger CapG enrichment in the nucleus. Lower panels: MDA-MB-231 cells were transfected with CAPNb4-EGFP coupled to the SV40 large T-antigen NLS, redirecting endogenous CapG (red) nearly completely to the nucleus. DAPI staining of the nucleus is shown in blue at right. Bar, 10 μm. Quantification of this experiment is shown in the histogram at right. The nuclear enrichment of CapG was evaluated by determining the ratio of cytoplasmic versus nuclear CapG. Statistics were performed on 50 cells per condition, with the EGFP (control) condition as reference (***P < 0.001).