Fig. 4. Characterization of anti-MACF1 (CU119) antibody. (A) Detection of the full-length plakin domain of MACF1 proteins by CU119 antibody. COS7 cells were transfected with plasmid pFLAG-MACF1-plakin and cell lysate was resolved by SDS-PAGE. The transblot was then probed with mouse monoclonal anti-FLAG antibody (M2) and anti-MACF1-plakin antibody (CU119). The expressed full-length MACF1-Plakin protein (FL-plakin) can be detected by both M2 and CU119 antibodies, but not in mock-transfected control (C). (B) Detection of endogenous MACF1 proteins by CU119 antibody in CAD cells. Neuronal CAD cells were lysed and resolved by SDS-PAGE, and the transblot was probed with CU119 antibody. MACF1 proteins can be detected in CAD cells (lane 2), and this signal can be completely abolished by pretreatment of CU119 antibody with antigen (lane 1). (C) Specificity of CU119 antibody revealed by the detection of MACF1 proteins in siRNA-treated cells. H460 cells were transfected with MACF siRNA vector (MACF-siRNA) that was designed to knockdown MACF1, or control vector (Control) for 72 hours. After SDS-PAGE, the transblot was probed for either MACF1 (top panel) or ß-tubulin (loading control; lower panel). MACF1 proteins in cells transfected with MACF siRNA vector (MACF siRNA) were reduced compared to cells transfected with the control vector (Control). (D) Localization of MACF1 in H460 cells. H460 cells were stained for MACF1 (left panel; CU119 antibody that recognizes all isoforms; green) and ß-tubulin (middle panel; red). MACF1 protein was found around the nuclei, dotted in the cytoplasm and decorating microtubules (right panel; merged picture).