Anti postn

Cells were lysed with RIPA buffer containing a phosphatase and protease inhibitor cocktail. Cell protein (30–40 μg) was used for Western blotting analysis as described [1 (link), 24 (link)]. C28/I2 cells seeded at 80% confluency were grown overnight in DMEM supplemented with 10% FBS, and then starved for 24 h in serum-free medium. The cells were then transfected with DDR1-Fc and/or Postn cDNA as described [1 (link)]. After 24–48 h, the cells were washed with cold PBS and lysed with RIPA buffer containing phosphatase and protease inhibitors. One milligram of cell extract protein was mixed with 10 μl Protein A & G Sepharose (Sigma-Aldrich) and 10 μl anti-Postn (Sigma-Aldrich) or anti-DDR1 antibodies (Cell Signaling), or with control IgG for 24 h at 4° C. Antigen-antibody complexes were then analyzed by Western blotting as described above.

Cell lysates were prepared in radio-immunoprecipitation buffer, separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes. Following blocking, the membranes were incubated overnight at 4 °C with anti-POSTN (1:500), anti-PDPN (1:1000), anti-calumenin (1:500), anti-α-SMA (1:500), or anti-β-actin (1:10000; Sigma) antibodies. The membranes were washed again and incubated for 1 h at RT with secondary antibodies. The antigen was then detected using the ImmunoStar Basic Chemiluminescence Reagent (WAKO Pure Chemical Industries Ltd, Osaka, Japan), according to the manufacturer’s instructions. Protein bands were captured by ChemiDoc Touch (Bio-Rad, Hercules, CA, USA). Signal intensities were quantified using ImageLab (6.0 version) software (Bio-Rad), normalized to their loading control β-actin, and expressed as fold change compared with controls.

After treatment of IL-6 and/or blockers of signaling pathways. POSTN protein was analyzed from the collected cell lysate samples by Western blot analysis. Briefly, the cell lysate samples were homogenized in ice-cold modified RIPA buffer (50 mmol/l Tris–HCl, pH 7.4, 1% NP-40, 0.25% deoxycholic acid, 0.15 mol/l NaCl, 1 mmol/l EDTA, 1 mmol/l PMSF/NaF/sodium orthovanadate, and protease inhibitors cocktail). Supernatants were collected after centrifugation. The proteins were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane at 200 mA for 2 h at room temperature. The blot was blocked with 5% non-fat dry milk suspended in 1 × phosphate-buffered saline, 0.1% Tween® 20 detergent for 1 h at room temperature, and then overnight at 4 °C with the following primary antibodies: anti-POSTN (1:500, Sigma-Aldrich, St Louis, MO) and anti-β-actin (1:30,000, Sigma-Aldrich, St Louis, MO). The membranes were washed and incubated in blocking solution with the appropriate HRP-conjugated secondary antibody for 2 h at room temperature (1:2000, Sigma-Aldrich, St Louis, MO). The signals were visualized directly using a UVP BioSpectrum ® 810 Imaging System.