In vivo ms fx pro

Cells were harvested 24 hrs after seeding on PUR substrates in a radioimmunoprecipitation buffer containing a cocktail of protease and phosphatase inhibitors (Pierce). Equal protein concentrations were prepared for loading with NuPAGE sample buffer (Life Technologies) and separated on a 10% SDS-PAGE gel (BioRad). Proteins were transferred to a PVDF membrane and blocked with 5% BSA in TBS containing 0.1% Tween-20 for 1h at room temperature, followed by incubation with either phospho-p38MAPK (1:1000, Cell Signalling), p38MAPK (1:1000, Cell Signalling), phospho-Smad2/3 (1:1000, Cell Signalling) or Smad2/3 (1:1000, Cell Signalling) antibodies overnight at 4°C. After washing, membranes were blotted with anti-rabbit IgG (1:2000, SantaCruz), and bands were detected by enhanced chemiluminescence using an In-Vivo MS FX Pro (Bruker). Membranes were then stripped and reprobed using an antibody for β-actin (1:5000, Sigma) as a loading control. Phosphorylated events were quantified by normalizing the band intensity of phosphorylated protein to total protein. Analysis was performed using Image J software.

The R7 murine breast cancer cell line was derived from a mammary tumor from a transgenic MMTV-Ron mouse.(Zinser et al., 2006 (link)) For our in vivo experiments, 150,000 R7 cells were orthotopically injected into the inguinal mammary fat pads of syngeneic FVB female mice following protocols previously described.(Wagh et al., 2011 (link)) Tumor formation was examined by manual palpation after 10-day post-injection. Alfalfa-free diet was fed to the mice upon identification of palpable tumors. To label PLA with Cy5.5, Cy5.5-NH₂ was conjugated to PLA-COOH through the carbodiimide-mediated coupling reaction. Near infrared fluorescence (NIFR) NPs were prepared by blending 10 wt% of PLA-Cy5.5 and 90 wt% PLA-PEG before nanoprecipitation. NPs were then injected through the tail vein, and mice were imaged 1 hour, 4 hours, and 24 hours after the injection using a fluorescent imaging system (Bruker In-Vivo MS FX PRO). All animal treatments and surgical procedures followed approved protocols and were performed in accordance with the Institutional Animal Care and Use Committee of the University of Cincinnati.

To investigate association of membrane proteins, cells were lysed using a 1% Nonidet-P40 buffer containing a cocktail of protease and phosphatase inhibitors (Pierce). 400μg total protein lysate per tube was incubated overnight at 4°C under gentle end-over-end mixing with anti-TGF-βRII (1 μg, Santa Cruz). Subsequently, the immune complex was captured with protein A/G agarose resin, thoroughly washed with lysis buffer and eluted with non-reducing sample buffer. Proteins were then separated on a 10% SDS-PAGE gel (BioRad) and transferred to a PVDF membrane. Following blocking with 5% milk in TBS with 0.1% Tween-20, membranes were incubated with anti-Iβ3 (Santa Cruz, 1:1000) or anti-TGF-β RII (Santa Cruz, 1:1000) at 4°C overnight. After washing, membranes were blotted with anti-rabbit or anti-mouse IgG (1:5000 Santa Cruz), and bands were detected by enhanced chemiluminescence using an In Vivo MS FX Pro (Bruker). The Iβ3 and TGF-βRII co-precipitation was quantified by normalizing the band intensity of Iβ3 pulled down with TGF-βRII to total protein (anti-TGF-βRII). Analysis was performed using Image J software.

Mice limbs were dissected, soft tissues were removed and fixed in 4% paraformaldehyde for 48 hours at 4ºC and then decalcified in 0.5M EDTA (PH 8.0) at 4ºC on a shaker for periods ranging from 7 to 14 days. Complete decalcification was confirmed by digital radiography (In Vivo MS FX PRO, Bruker Co). The tissues were cryoprotected in 15% sucrose/PBS for 1hr and 30% overnight at 4ºC and then placed in 30% sucrose/PBS:OCT (1:1) solution for 1hr. Samples were embedded in Tissue-Tek O.C.T. compound (Sakura, 4583) and transferred to dry ice to solidify the compound. Embedded samples were cryosectioned at 10 µm using a cryostat (OTF 5000, Bright Instrument Co Ltd.). Images were taken with Nikon 80i Upright microscope (Nikon Co.). Images of some optic fields were taken using blue and red fluorescence filters, and merged with MetaMorph Software (Molecular Devices LLC.).