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Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk
Article first published online: 1 OCT 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Volume 13, Issue 21, pages 3189–3204, November 2013
How to Cite
Janjanam, J., Jamwal, M., Singh, S., Kumar, S., Panigrahi, A. K., Hariprasad, G., Jena, M. K., Anand, V., Kumar, S., Kaushik, J. K., Dang, A. K., Mukesh, M., Mishra, B. P., Srinivasan, A., Reddy, V. S. and Mohanty, A. K. (2013), Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk. Proteomics, 13: 3189–3204. doi: 10.1002/pmic.201300031
- Issue published online: 4 NOV 2013
- Article first published online: 1 OCT 2013
- Accepted manuscript online: 9 SEP 2013 02:01AM EST
- Manuscript Accepted: 9 AUG 2013
- Manuscript Revised: 5 AUG 2013
- Manuscript Received: 19 JAN 2013
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Figure S1. Characterization of purified epithelial cells using RT-PCR, qRT-PCR and western blot analysis: A. RT-PCR analysis for IL-8, CD45, CD163, CD19 and CD4. Lane M: 100 bp ladder, in A(I) lanes A and D: skin fibroblasts (negative control), lanes B and E: somatic cells (positive control) and lanes C and F: MECs. In A(II) lane A: somatic cells, lane B: MECs, lane C: skin fibroblasts. In A(III) lanes A and B: skin fibroblasts, lanes C and E: somatic cells, lanes D and F: MECs. B. RT-PCR analysis for β-casein (I), Cytokeratin8 (II) and SMA (III); lane M:100 bp ladder; lane A: Somatic cells (Positive control); lane B: MECs; lane C: Culture MECs and lane D: Skin fibroblasts (Negative control) andβ-actin was used as loading control.. C. Relative expression of α-lactalbumin in MEC and somatic cells: α-LA is specific to MEC and the expression of α-LA in purified MEC was observed to be 112 fold higher than the expression in total somatic cells. D. Western blot analysis of CD45: Detection of CD-45 using anti- CD45. M: marker, L: leukocytes (positive Control), MEC: Mammary Epithelial Cells, F: Fibroblast Cells (negative control). Absence of CD45 in MEC lane suggests that they were free from immune cells. E. Western blot analysis of casein in MECs isolated from milk. Detection of casein using anti-casein antibodies, Lane 1, 2: MEC lysate isolated from milk, Lane 3, 4: total somatic cells and Lane 5, 6: unbound somatic cells, M: standard β-casein (Sigma).
Figure S2. RT-PCR analysis for Neutrophil cytosolic factor (NCF) and Bactericidal permeability increasing protein (BPI) (I); M: 100 bp ladder; A and E: Skin fibroblasts (Negative control- NCF and BPI); B and F: Somatic cells (Positive control- NCF and BPI); C and G: Mammary Epithelial Cells (NCF and BPI); D and H: Culture Mammary Epithelial Cells (NCF and BPI); Neutrophil Elastase(II); A: Skin fibroblasts (Negative control); B: Somatic cells (Positive control); C: Mammary Epithelial Cells; D: Culture Mammary Epithelial Cells and CD14(III); A: Somatic cells (Positive control); B: Mammary Epithelial Cells; C: Culture Mammary Epithelial Cells and D: Skin fibroblasts (Negative control); β-actin was used as loading control.
Table S1. Characteristics of primer pairs used for PCR amplification of MEC specific genes like cytokeratin 8, α-lactalbumin, smooth muscle antigen, β-casien, β-Actin, IL-8, CD45,CD19, CD4,CD163, CD14, neutophill cytosolic factor, neutophill elastase, bactericidal permeability increasing protein.
Table S2. List of proteins identified from 2 DE reference map
Table S3. List of proteins identified using GeLC-MS/MS
Table S4. MEC proteins identified by 2-DE-MALDI-TOF-TOF and GeLC-MS/MS
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