Epithelial cell lysates were prepared using Cytobuster Protein Extraction Reagent (71009, EMD Millipore, Watford, UK) with protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets, Roche, Burgess Hill, UK). Stromal cell lysates were prepared in a 1% TX-100 lysis buffer containing protease inhibitors. SDS-PAGE and western blotting were as described previously (Rumsby et al, 2011 (link)). Primary antibodies were an anti-PLD1 rabbit polyclonal (sc25512, Santa Cruz, Insight Biotechnology, Wembley, UK, 1 : 750), and a rabbit anti-GAPDH polyclonal (Abcam, Cambridge, UK, ab9485, 1 : 20 000). The secondary antibody was HRP-linked anti-rabbit IgG (7074S, Cell Signalling, New England Biolabs Ltd, UK, 1 : 6000). A kaleidoscope protein ladder (Bio-Rad, Watford, UK, Cat No 1610375) was used throughout.
Protocol detail
Western Blot Analysis of PLD1 in Epithelial and Stromal Cells
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Cytobuster Protein Extraction Reagent
Protease Inhibitor Cocktail Tablets
sc25512
ab9485
HRP-linked anti-rabbit IgG (7074S,
kaleidoscope protein ladder
Anti igg
Antibodies
Antibody
Buffer
Edta
Epithelial cell
Gapdh
Pld1
Protease inhibitor
Protease inhibitors 1
Protein
Rabbit
Sds page
Stromal cell
Tx 100
Corresponding Organization : University of York
Other organizations : Queen Mary University of London
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Variable analysis
independent variables
- Cell type (epithelial vs stromal)
- Protein expression levels of PLD1 and GAPDH
- Lysis buffer composition (Cytobuster Protein Extraction Reagent with protease inhibitors for epithelial cells, 1% TX-100 lysis buffer with protease inhibitors for stromal cells)
- SDS-PAGE and western blotting protocol (as described previously in Rumsby et al., 2011)
- Primary antibodies (anti-PLD1 rabbit polyclonal, rabbit anti-GAPDH polyclonal)
- Secondary antibody (HRP-linked anti-rabbit IgG)
- Protein ladder (Kaleidoscope protein ladder)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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