RTM28-transduced RDEB keratinocytes were grown to confluence in conditioned medium. The medium was changed containing 50 μg ml−1 ascorbic acid, and the cultures were maintained for an additional 48 h. Cells were harvested and lysed in 50–100 μl RIPA lysis buffer (Santa Cruz Biotechnology, Dallas, TX, USA). After 20 min of centrifugation at 4 °C, 10–20 μg protein samples were mixed with 4x sodium dodecyl sulphate-polyacrylamide gel electrophoresis sample buffer (0.25 m Tris-HCl; 10% sodium dodecyl sulphate; 40% glycerol; 0.008% bromphenol blue; 2.86 m β-mercaptoethanol; pH 6.8), denatured for 5 min at 95 °C and loaded onto a Bolt 8% Bis-Tris Plus Gel (Novex, Life Technologies, Carlsbad, CA, USA). Western blot analysis was performed as described previously.23 (link), 40 (link) For detection of type VII collagen, the primary antibody rabbit anti-type VII collagen (kindly provided by Dr Alexander Nyström) was added to the blocking solution at a dilution of 1:3000 and incubated overnight at 4 °C, followed by incubation with the secondary antibody, anti-rabbit horse radish peroxidase (Dako, Glostrup, Denmark) 1:1000 in blocking buffer (Roche) for 1 h at room temperature.
For the detection of FLAG-tagged fusion proteins, blots were incubated with the anti-FLAG M2mAb (Sigma-Aldrich) diluted at 1:1000 in blocking buffer (Roche).
For the detection of FLAG-tagged fusion proteins, blots were incubated with the anti-FLAG M2mAb (Sigma-Aldrich) diluted at 1:1000 in blocking buffer (Roche).
