1. BPAG1-e Restricts Keratinocyte Migration through Control of Adhesion Stability 
Magdalene Michael, Rumena Begum, Kenneth Fong, Celine Pourreyrone, Andrew P. South, John A. McGrath, Maddy Parsons 
Journal of Investigative Dermatology 
Volume 134, Issue 3, Pages (March 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Loss of bullous pemphigoid antigen 1 (BPAG1-e) results in decreased adhesion and increased migration speeds. (a, b) Wild-type (WT) and dystonin (DST) mutant cells were plated and fixed after 30, 60, and 120 minutes. (a) Immunofluorescence images of adherent cells stained with phalloidin A568. Scale bar= 100 μm. (b) Quantification of number of adherent cells at indicated time points. (c) Quantification of cell detachment following trypsin treatment. (d–f) Analysis of random migration of WT and DST mutant keratinocytes. (d) Migration tracks of keratinocytes were analyzed for (e) speed and (f) persistence. For all quantification, data represent three independent experiments, error bars=SEM; *P<0.05, **P<0.01, and ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Knockdown (KD) of bullous pemphigoid antigen 1 (BPAG1-e) does not recapitulate dystonin (DST) mutant adhesion and migration defects. (a) Western blot analysis of BPAG1-e protein levels in control and BPAG1-e KD keratinocytes. (b, c) Adhesion of BPAG1-e KD cells following 30, 60, and 120 minutes of plating. (b) Immunofluorescence images of cells stained with phalloidin A568. Scale bar=100 μm. (c) Quantification of number of adherent cells. (d–f) Random migration analysis of control and BPAG1-e KD cells. (d) Migration tracks of keratinocytes were analyzed for (e) speed and (f) persistence. (g) Western blot analysis of control and BPAG1-e KD keratinocytes with keratin 14 antibodies and (h) assessment of KRT14 mRNA levels by reverse-transcriptase–PCR (RT-PCR), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. (i) FACS analysis of surface levels of β4, β1 total, and β1 active (A) integrins in control and BPAG1-e KD cells. For all quantification, data represent three independent experiments; error bars=SEM; *P<0.05.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Keratin 14 (K14) upregulation does not contribute to adhesion defect in dystonin (DST) mutant cells. (a) Immunofluoresence of wild-type (WT) and DST keratinocytes with K14 antibodies and phalloidin A568. Scale bar=10 μm. (b) Cell area measurements of WT and DST keratinocytes monolayers. (c, e, f) Western blot analysis of (c) K14 and (e) K5 protein expression in WT and DST mutant cell lines and (f) K14 expression in WT, DST1, and DST1 cells stably expressing two different K14 short hairpin RNA (shRNA) (K14KD1 and K14KD2). Hsc70 was used as a loading control. (d) Assessment of KRT14 mRNA expression by reverse-transcriptase–PCR (RT-PCR) in WT and DST1 cells, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. (g) Adhesion analysis of WT, DST1, and DST1 expression K14KD2 shRNA after 30, 60, and 120 minutes of plating. For all quantification, data represent three independent experiments, error bars=SEM; *P<0.05, **P<0.01, and ***P< AU, arbitrary units.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Dystonin (DST) mutation results in an integrin switch in keratinocytes. (a) Immunostaining of wild-type (WT) and DST mutant skin sections for β4 and α6 integrin. Scale bar=30 μm. (b) Western blot analysis of β4 integrin and β1 integrin in WT and DST mutant cell lysates. Hsc70 was used as a loading control. (c, d) FACS analysis of (c) β4 and α6 integrin surface levels and (d) β1 integrin total and active (A) surface levels in WT and DST mutant cells. For all quantification, data represent three independent experiments, error bars=SEM; *P<0.05, **P<0.01.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions