Las 3000 device

Cells were lysed in RIPA Buffer (Sigma-Aldrich) with protease inhibitor cocktail (Sigma-Aldrich). Homogenates were centrifuged (15000 × g, 30 min, 4°C) and the protein concentration of the supernatant was calculated using a DC protein assay (Bio-Rad, Hercules, CA). The lysates were boiled with 5×sample buffer with 2-mercaptoehanol and finally subjected to SDS-PAGE (7.5%). The blots were blocked using blocking buffer (Beacle, Kyoto, Japan) for 12 hr at 4°C, and then incubated with anti-TASK1 or anti-TALK2 antibodies for 12 hr at 4°C. After washing, the blots were incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG (Chemicon International, Temecula, CA). An enhanced chemiluminescence detection system (GE Healthcare Biosciences, Piscataway, NJ) was used to obtain images of the bound antibody. The resulting images were analyzed using a LAS-3000 device (Fujifilm, Tokyo, Japan).

BM-MSCs seeded in 3D gelatin scaffolds were cultured for 24 hours or 10 days. Cells were lysed with 75 μl of lysis buffer (PBS, 1% Triton X-100, 100 mM PMSF, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 mM of sodium orthovanadate, 10 mM NaF and 10 mM β-glycerophosphate) for one hour at 4°C. Thirty micrograms of protein samples were subjected to SDS-PAGE and immunoblotting. Membranes were incubated with different antibodies: pGSK3α/β Ser9/21 (9331S), pSMAD1/5/8 Ser465/467 (9511S) and pS6 Ser235/236 (2211) and pp38 Thr180/Tyr182 (9211S) from Cell Signaling Technology, pErk1/2 (M5670) from Sigma, β-catenin (610154) from BD Transduction Laboratories and α-tubulin (T6199) from Sigma, all diluted to a ratio of 1:1000. Horseradish peroxidase-conjugated secondary antibodies were used, followed by incubation with EZ-ECL reagent (Biological Industries). A chemiluminescent image of the immunoblots was captured with a Fujifilm LAS 3000 device.

Co-IP was performed using the Pierce Co-Immunoprecipitation Kit (number 26149) according to the experimental manual supplied by Thermo Scientific (Waltham, MA) (22 (link)). The rat mesenteric artery was lysed in IP lysis/wash buffer with protease inhibitor mixture (Sigma). Precleared whole lysates were incubated with AminoLinkPlus Coupling Resin or Control Agarose Resin slurry with which 10 μg of anti-Cav1 was immobilized, washed with IP lysis/wash buffer, eluted with elution buffer, and subjected to SDS-PAGE (10%). In Western blot analysis, 30 μg of total protein was applied to the gels. The blots were incubated with anti-JP2 antibodies and then incubated with anti-goat horseradish peroxidase-conjugated IgG (Chemicon International, Temecula, CA). Images were obtained using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) and analyzed by a LAS-3000 device (Fujifilm, Tokyo, Japan). Full western blot images are presented in Fig. S8.

Co-IP procedures were carried out using a Pierce Co-Immunoprecipitation Kit according to the experimental manual supplied by Thermo Scientific [19 (link)]. Briefly, HEK293 cells were lysed in IP Lysis/Wash Buffer (0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol; pH 7.4) with protease inhibitor cocktail (Sigma-Aldrich). Homogenates were centrifuged (15000 × g, 30 min, 4°C) and supernatant was precleared with control resin (1 h, 4°C). Precleared lysates (~ 0.5 mg of protein) were incubated with AminoLink Plus Coupling Resin, with which 10 μg anti-GFP antibody (mFX73, Wako) was immobilized (overnight, 4°C). The incubated lysates were washed with IP Lysis/Wash Buffer, eluted with Elution Buffer, boiled with 5× sample buffer with 2-mercaptoehanol and finally subjected to SDS-PAGE (10%). The blots were incubated with anti-TASK1 antibody and then incubated with anti-rabbit horseradish peroxidase-conjugated IgG (Chemicon International, Temecula, CA). An enhanced chemiluminescence detection system (GE Healthcare Biosciences, Piscataway, NJ) was used for getting images of the bound antibody. Resulting images were analyzed by a LAS-3000 device (Fujifilm, Tokyo, Japan).