Completetm tablets

To identify the fraction of receptor protein inserted in the plasma membrane, surface proteins were tagged with biotinylated ConA and isolated by streptavidin-sepharose-mediated precipitation of the biotinyl-ConA-protein complex as previously described by Strutz-Seebohm et al. (2003 (link)). For determination of cytosolic proteins, intact oocytes were homogenized with a teflon pestle in H-buffer (25 μl/oocyte; 100 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride plus a mixture of proteinase inhibitors; Complete^TM tablets, Roche Applied Science, Penzberg, Germany). After centrifugation for 2 min at 16,000×g, the supernatants were supplemented with 9 μl of SDS-PAGE loading buffer (0.8 M ß-mercaptoethanol, 6% SDS, 20% glycerol, 25 mM Tris-HCl, pH 6.8, 0.1% bromphenol blue).

Primary HSCs were cultured in DMEM (GIBCO BRL) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, glutamine, 10% fetal bovine serum (GIBCO BRL) and labeled with either l-arginine and l-lysine, l-[U-¹³C₆,¹⁴N₄] arginine and l-[²H₄]lysine, or l-[U-¹³C₆,¹⁵N₄]arginine and l-[U-¹³C₆,¹⁵N₂]lysine (Cambridge Isotope Laboratories, Andover, MA, USA; Sigma-Aldrich) (2*15-cm cell culture dishes per condition; ~95% confluent). Cells were washed three times in PBS before they were treated with DMSO or 20 μM DHA following 20 ng/ml PDGF-BB treatment for indicated hours. All treatments were carried out at 37 °C. After drug treatment, the cells were scraped in cell scraping buffer (0.25 M sucrose, 1 mM sodium ortho-vanadate, 5 mM NaF, 5 mM β-glycerophosphate, and protease inhibitor mixture (Complete TM tablets, Roche Diagnostics)) and normalized by cell counting. Mixed cells were centrifuged for 5 min at 1800 rpm and lysed in 6 M urea and 2 M thiourea, and 2% Benzonase (Merck) was added before samples were concentrated on spin tubes (cutoff, 500 Da). Protein mixtures were separated by SDS-PAGE (4–12% bis-Tris gra-dient gel, NuPAGE, Invitrogen). Gel lanes were cut into 15 slices, and samples were in-gel digested, and resulting peptide mixtures were STAGE-tipped. Relative quantification and identification of peptides were analyzed by LC–MS/MS as described previously.^{39 (link)}

Frozen cell pellets were homogenized in cold RIPA buffer (Sigma R0278, Saint Quentin Fallavier, France) containing protease inhibitors (cOmplete^TM Tablets, Roche 4693159001, Merck, Saint Quentin Fallavier, France) for 1 h. An equal amount of 10 µg protein per sample was used. Western Blot migration, transfer, incubation, revelation, and quantification were performed as previously described using mouse anti-β-actin (1:20,000, Sigma A5316, Saint Quentin Fallavier, France), mouse anti-CNPase (1:500, Merck-Millipore MAB326, Guyancourt, France), and HRP-conjugate goat anti-mouse IgG (1:2000, Sigma 12-349, Saint Quentin Fallavier, France) antibodies [16 (link)]. Actin was used as loading control.