mouse monoclonal antibody against igf ii  (R&D Systems)

Journal: Molecular cancer research : MCR

Article Title: Intrinsic Resistance to Cixutumumab is Conferred by Distinct Isoforms of the Insulin Receptor

doi: 10.1158/1541-7786.MCR-15-0279

Figure Lengend Snippet: Insulin-like growth factor II (IGF-II) mediates resistance to cixutumumab in tumor cells over-expressing IR-B. Clonogenic assay in A549 cells that overexpress IR-B (A) in the presence or absence of cixutumumab and/or anti-IGF-II mAb. Represented are mean values +/− SEM, expressed as percentage of colony formation relative to control treated cells. Statistical significance was determined by one-way ANOVA followed by Tukey’s posthoc analysis. Statistically significant difference vs control treated cells (a), cixutumumab-treated cells (b), anti-IGF-II treated cells (c). (B, C) Western blot analysis of signal transduction in A549-IR-B cells treated with IGF-II (25 nM), in the presence or absence of cixutumumab and linsitinib. Serum-starved cells were pretreated with cixutumumab (100 nM) or linsitinib (0.01, 0.1 or 1 uM) for 15 min and 2 h, respectively, followed by incubation with rhIGF-II (25 nM) for 10 minutes. Proteins (15–20 ug) extracted from cultured cells were size-fractionated by SDS-PAGE and immunoblotted with anti-phospho-IRβY1150/51/IGF-IRβY1135/36, anti-phospho-AktS473 and anti-phospho-p42/p44 MAPKT202/Y204 antibodies. Total level of proteins was demonstrated by immunoblotting with antibodies directed against total Akt and p42/p44 MAPK. (D) MCF-7 and A549 cells with or without IR-A or IRB overexpression were treated with increasing concentrations of linsitinib (0.00015-10 uM) for 72 h, and tumor cell viability was quantified by CellTiter-Glo assay. The results are expressed as % of inhibition of tumor cell viability.

Article Snippet: Antibodies The following antibodies were purchased from commercial sources as indicated: mouse monoclonal antibodies against Akt (#2920) and p42/p44 MAPK (Erk1/2) (#9107), rabbit monoclonal antibodies against phospho-Akt S473 (#4060) and phospho-IGF-IR beta Y1135/1136 / Insulin Receptor beta Y1150/1151 (#3024), rabbit polyclonal antibody against phospho-p42/p44 MAPK T202/Y204 (Erk1/2) (#9101) (Cell Signalling, Beverly, MA, USA); mouse monoclonal antibody against IGF-IR (#MS-641-P) and Insulin Receptor (#MS-632-P) (Thermo Fisher Scientific, Fremont, CA, USA); rabbit polyclonal Insulin Receptor (#sc-711) and (#sc-7953) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal antibody against IGF-II (MAB292) and goat F(ab') 2 anti-mouse IgG-phycoerythrin (#F0102B) (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-mouse IRDye 680 conjugated (#926–32220) and anti-rabbit IRDye 800 conjugated (#926–32211) (LI-COR Biosciences, Lincoln, Nebraska, USA).

Techniques: Expressing, Clonogenic Assay, Control, Western Blot, Transduction, Incubation, Cell Culture, SDS Page, Over Expression, Glo Assay, Inhibition

Journal: The Prostate

Article Title: Exogenous SPARC suppresses proliferation and migration of prostate cancer by interacting with integrin β1.

doi: 10.1002/pros.22664

Figure Lengend Snippet: Fig. 1. Immunohistochemical stainingofSPARC onprostate tissue.CommercialPCa tissuemicroarray (TMA) slides (A^H) were deparaffi- nized in xylene and rehydrated in graded alcohols and processed as described method.TMAs with 96 specimens and tissue sections were immunostained with SPARC antibody.Representative examples of photomicrographs are showing SPARC expression in the non-neoplastic prostateandPCa.A:non-neoplastic fromE.B:non-neoplastic fromF.C:non-neoplastic fromG.D:non-neoplastic fromH.E:Gleasonscore7. F:Gleasonscore7.G:Gleasonscore8.H:Gleasonscore9(originalmagnification40and100).

Article Snippet: After blocking endogenous peroxidase activity using 3% hydrogen peroxide for 10 min, the sections were incubated with rabbit monoclonal antibodies against SPARC (R&D system) at a dilution of 5 mg/200 ml at room temperature for 60 min. After the primary antibodies reaction, sections were washed with PBS slowly and followed by peroxidase labeled secondary antibody (a mixture of rabbit and mouse antibodies) combined with dextranpolymers.

Techniques: Immunohistochemical staining, Expressing

Journal: The Prostate

Article Title: Exogenous SPARC suppresses proliferation and migration of prostate cancer by interacting with integrin β1.

doi: 10.1002/pros.22664

Figure Lengend Snippet: Fig. 2. Expression level of SPARC in PCa cells, PrSC, and PCaSCs. A: Expression levels of SPARC mRNAwere measured in PCa cell lines (LNCaP, DU145, and PC-3) and stromal cell lines (PCaSC-5, PCaSC-8, PrSC) by RT-PCR.B: Whole cell lysate from each cells were also sub- jectedtoWesternblotanalysis andnormalizedasaratiousingb-actin.C:Eachcell(4 105)wasculturedinserum-freemediumfor36 hr and SPARCproteinconcentrationsin themediumweremeasuredbyELISAassay.ODvalues obtainedbyELISAreader at450 nmare expressedas means SDfromfourreplicate experiments.Cells (1 106 cells) were culturedwith 0.2% FBSfor 2 days and the supernateswereincubated withcommerciallyavailable arraymembrane.D: ComparisonofSecretedSPARCinPrSC and PCaSC-5.Imageswere collectedusing achemi- luminescenceimaging system after cytokine array. Srepresents secreted SPARC (top).The data on thebottomrepresentsrelative amountof secretedSPARC.

Article Snippet: After blocking endogenous peroxidase activity using 3% hydrogen peroxide for 10 min, the sections were incubated with rabbit monoclonal antibodies against SPARC (R&D system) at a dilution of 5 mg/200 ml at room temperature for 60 min. After the primary antibodies reaction, sections were washed with PBS slowly and followed by peroxidase labeled secondary antibody (a mixture of rabbit and mouse antibodies) combined with dextranpolymers.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Prostate

Article Title: Exogenous SPARC suppresses proliferation and migration of prostate cancer by interacting with integrin β1.

doi: 10.1002/pros.22664

Figure Lengend Snippet: Fig. 3. Inactivation of AKT by exogenous SPARC. A: After LNCaP, DU145, and PC-3 cells (4 105) were treated with vehicle (medium) or0.5,1.0 mg/mlSPARCfor2days,whole celllysatefromeach samplewas also subjectedtoWesternblotanalysis.B:LNCaP,DU145,andPC-3 cells were coculturedwithvehicle,PrSC,PCaSC-6, and PCaSC-7 for 48 hr in a transwellchamber asillustrated transwellcoculture systems. The celllysates fromPCacellswere subjectedtowesternblotanalysiswithanti-phospho-AKTandanti-AKTantibody(Leftpanel).Rightpanel representsrelative changes ofphospho-AKTexpression.C:Knockdown ofSPARC expressionin PrSC by SPARC siRNAtransfection.Expres- sionofSPARCmRNAwasdetectedbyRT-PCR(Top).After transfectionofSPARCsiRNA(4 105 cells),SPARCconcentrationsinthemedium weremeasuredbyELISA(Bottom).D:LNCaP,DU145,andPC-3werecoculturedwith20 nmol/L SPARC siRNAtransfectedPrSCfor48 hrin a transwellchamber.The celllysateswithanequalamountofproteinsincellswere subjectedtowesternblotwithanti-phospho-AKTandanti- AKTantibody.

Article Snippet: After blocking endogenous peroxidase activity using 3% hydrogen peroxide for 10 min, the sections were incubated with rabbit monoclonal antibodies against SPARC (R&D system) at a dilution of 5 mg/200 ml at room temperature for 60 min. After the primary antibodies reaction, sections were washed with PBS slowly and followed by peroxidase labeled secondary antibody (a mixture of rabbit and mouse antibodies) combined with dextranpolymers.

Techniques: Knockdown

Journal: The Prostate

Article Title: Exogenous SPARC suppresses proliferation and migration of prostate cancer by interacting with integrin β1.

doi: 10.1002/pros.22664

Figure Lengend Snippet: Fig. 4. Interaction of SPARC with integrin b1. A: After LNCaP, DU145,andPC-3cells(4 105)were treatedwithvehicle or0.5,1.0, and2.0 mg/mlSPARCfor2days,the expressionlevelsofintegrina1, a2, b1, and b2 mRNA were measured by RT-PCR. B: After cells were treated with 0, 0.5,1.0,1.5, and 2.0 mg/ml SPARC for 2 days, whole cell lysate from each sample were subjected to immunopre- cipitationwith anti-SPARC (source: rabbit) and anti-integrin b1.To- tal protein used for immunoprecipitation was confirmed with an anti-rabbit antibody as precipitated anti-SPARC antibody source wasrabbit.C:Cellswere treatedwith1.0 mg/mlSPARCfor2daysin presence or absence of an isotype control or integrin b1^blocking antibody (10 mg/ml). Immunoprecipitation was conducted with anti-SPARC and the precipitated proteins revealed with and anti- integrin b antibodies.Whole cell lysate from each sample was also subjected to western blot analysis with anti-phospho-AKT and anti-AKTantibody.

Article Snippet: After blocking endogenous peroxidase activity using 3% hydrogen peroxide for 10 min, the sections were incubated with rabbit monoclonal antibodies against SPARC (R&D system) at a dilution of 5 mg/200 ml at room temperature for 60 min. After the primary antibodies reaction, sections were washed with PBS slowly and followed by peroxidase labeled secondary antibody (a mixture of rabbit and mouse antibodies) combined with dextranpolymers.

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Control, Blocking Assay, Western Blot

Journal: The Prostate

Article Title: Exogenous SPARC suppresses proliferation and migration of prostate cancer by interacting with integrin β1.

doi: 10.1002/pros.22664

Figure Lengend Snippet: Fig. 5. Effectof exogenous SPARC onproliferation ofprostate cancercells.A:LNCaP,DU145, and PC-3 cells (3 104/well) were cultured with vehicle (media) or 1 mg/ml SPARC in 5% FBS-containing medium for 12, 24, 36, and 72 hr. Cell proliferation was determined with a colorimetric WST-1/ECS assay.Experiments were conducted with three experimental replicates. B: Cells (3 104/well) were treated with 1.0 mg/mlSPARCfor72 hrinpresenceorabsenceofanisotypecontrolorintegrinb1^blockingantibody(10 mg/ml).

Article Snippet: After blocking endogenous peroxidase activity using 3% hydrogen peroxide for 10 min, the sections were incubated with rabbit monoclonal antibodies against SPARC (R&D system) at a dilution of 5 mg/200 ml at room temperature for 60 min. After the primary antibodies reaction, sections were washed with PBS slowly and followed by peroxidase labeled secondary antibody (a mixture of rabbit and mouse antibodies) combined with dextranpolymers.

Techniques: Cell Culture

Journal: The Prostate

Article Title: Exogenous SPARC suppresses proliferation and migration of prostate cancer by interacting with integrin β1.

doi: 10.1002/pros.22664

Figure Lengend Snippet: Fig. 6. Effectof exogenous SPARC onmigration ofprostate cancercells.A:Representativephotographs ofmigratedcells thatreceivedei- thercontroltreatmentorexogenousSPARC.Cellswerewoundedandthentreatedwithvehicle(media)or1 mg/mlSPARCin5%FBS-contain- ing medium. Images are taken immediately after scratching the cultures (0 hr),12, and 36 hr later (original magnification, 40). B: LNCaP, DU145,andPC-3cellswereculturedunder vehicle or0.5,1.0,and2.0 mg/mlSPARCconditionsfor2days.Thecellswereincubatedonthe8 mm pore polycarbonate membrane in a transwell.C: Cells were treated with1.0 mg/ml SPARC for 2 days in presence or absence of an isotype controlorintegrinb1-blockingantibody(10 mg/ml).Cellmigrationwasassessedby transwellmigrationassay.

Article Snippet: After blocking endogenous peroxidase activity using 3% hydrogen peroxide for 10 min, the sections were incubated with rabbit monoclonal antibodies against SPARC (R&D system) at a dilution of 5 mg/200 ml at room temperature for 60 min. After the primary antibodies reaction, sections were washed with PBS slowly and followed by peroxidase labeled secondary antibody (a mixture of rabbit and mouse antibodies) combined with dextranpolymers.

Techniques: Membrane

Journal: The Prostate

Article Title: Exogenous SPARC suppresses proliferation and migration of prostate cancer by interacting with integrin β1.

doi: 10.1002/pros.22664

Figure Lengend Snippet: Fig. 7. Function of SPARC on prostate cancer. Mechanism of how exogenous SPARC from stromal cells effect on PCa cells growthandmigration.

Article Snippet: After blocking endogenous peroxidase activity using 3% hydrogen peroxide for 10 min, the sections were incubated with rabbit monoclonal antibodies against SPARC (R&D system) at a dilution of 5 mg/200 ml at room temperature for 60 min. After the primary antibodies reaction, sections were washed with PBS slowly and followed by peroxidase labeled secondary antibody (a mixture of rabbit and mouse antibodies) combined with dextranpolymers.

Techniques: