Journal: International Journal of Molecular Sciences
Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells
Figure Lengend Snippet: Effect of transforming growth factor β (TGFβ 2 ) on SK-1, phospho-Smad2, connective tissue growth factor (CTGF), and fibronectin expression in HK2 cells. ( A ) HK2 cells were taken unstimulated (−) or stimulated with 5 ng/mL of TGFβ 2 for the indicated time periods up to 24 h. Thereafter, cell lysates were prepared and equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and subjected to Western blotting using antibodies against SK-1 (1:2000 dilution), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000), phospho-Smad2 (1:1000), CTGF (1:500), and fibronectin (1:500). To detect secreted CTGF, equal volumes of supernatants of stimulated cells were taken for protein precipitation by trichloroacetic acid (TCA) and were then analysed the same way as described for lysates using the CTGF antibody. Band intensities were evaluated by a LICOR R imaging system. Data in ( B – F ) show the evaluated respective bands. Results are expressed as fold increase and are means ± S.D. ( n = 3); * p
Article Snippet: S1P and all C17-sphingolipid standards were from Avanti Polar Lipids Inc. (Alabaster, AL, USA); caged-S1P was from Enzo Life Sciences Inc. (Farmingdale, NY, USA); human TGF-β2 , TNFα, and IL-1β were from Peprotech (London, UK); BAF312 was from Selleck Chemicals Inc. (Houston, TX, USA); CYM5442, CYM5520, CYM5541, puromycin, the lentiviral particles of human Spns2 and SK-1 shRNA constructs (MISSION® shRNA), diamidine-phenylindole (DAPI), and the antibodies against α-tubulin and Spns2 (SAB2104271) were from Sigma Aldrich Chemikalien, (Buchs, Switzerland); antibodies against phospho-ERK1/2, phospho-Smad2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Danvers, MA, USA); antibodies against CTGF (L-20) and fibronectin (EP5) were from Santa Cruz (Heidelberg, Germany); the AQP1 antibody ( (B)) was from Abcam plc (Cambridge, UK); the human SK-1 antibody was generated and characterized, as previously described [ , ].
Techniques: Expressing, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Imaging