Ab6064

For protein isolation, cells were lysed with a radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, Heidelberg, Germany) 72 h after transfection. After lysis, cells were centrifuged, and the clarified supernatant was used for analysis. Western blot analysis was performed as previously described [5 (link)]. Type VII collagen was detected using a polyclonal anti-collagen type VII rabbit antibody or human-specific anti-collagen type VII antibody specific for the NC1 and NC2 domains [59 (link)], and as a loading control, a β-tubulin-specific antibody (ab6064; Abcam, Cambridge, UK) was used. Goat anti-rabbit HRP-labelled antibody (Dako, Santa Clara, CA, USA) was used as a secondary antibody. Protein bands were visualized with the Immobilon Western Chemiluminescent HRP Substrate (Merck, Darmstadt, Germany) using the ChemiDoc XRS Imager (BioRad, Hercules, CA, USA). Native C7 Western blots were performed as previously described [60 (link)].

Primary antibodies were purchased from Abcam (Cambridge, UK) and were used at following dilutions: β-tubulin (ab6064; 1:200), ABCa3 (ab24751; 1:300) and IQGAP1 (ab110203; 1:200). Fluorescently labelled secondary antibodies and alexa fluor 568 phalloidin were purchased from Molecular Probes (Invitrogen, Carlsbad, USA) and used at dilution of 1:400. Immunofluorescence was performed as described previously^{16 (link)}.