sheep polyclonal anti cd44 antibody  (R&D Systems)

Journal: Oncotarget

Article Title: The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma

doi: 10.18632/oncotarget.14092

Figure Lengend Snippet: ( A – B ) Knockdown of SPP1 expression in LN18 cells reduced formation of spheres. Human adherent LN18 glioma cells were transfected with siRNAs ON-Target PlusSMARTpool Human SPP1 or ON-TARGETplus Non-targeting Pool (Dharmacon) using 4D-nucleofector AMAXA. After 24 h cells were seeded at a low density (4000 cells/ml) onto non-adherent plates and cultured in a defined medium. Resulting spheres (100–200 μm in size) were counted 7 days after transfection (B). In parallel, the expression of SPP1 in parental cultures was determined by qPCR 48 h after transfection (A) to evaluate efficacy of gene silencing, n = 3. ( C – D ) Knockdown of GLI1 or CD44 expression in LN18 cells impaired sphere formation. LN18 glioma cells were transfected with ON-Target PlusSMARTpool Human GLI1 or CD44 or ON-TARGETplus Non-targeting Pool (Dharmacon) siRNAs using 4D-nucleofector AMAXA. The expression of GLI1 and CD44 in parental cultures was determined by qPCR 48 h after transfection; data are means ± s.d., n = 3 (C). After transfection cells were seeded at a low density onto plates dedicated for a cell suspension culture and cultured 7 days in a defined medium. The resulting spheres (100–200 μm in size) were counted (D). The results are expressed as mean ± s.d.; P values were considered significant when *P ≤ 0.05 and * *P ≤ 0.01, n = 3.

Article Snippet: CD44 was detected by Western blotting with polyclonal Sheep IgG anti-human CD44 (#AF3660, R&D System, Minneapolis, MN, USA, 1:500) followed by a rabbit Anti-Sheep IgG-HRP conjugate (#AP147P, Merck Millipore KGaA, Darmstadt, Germany, 1:5000).

Techniques: Knockdown, Expressing, Transfection, Cell Culture, Suspension

Journal: Oncotarget

Article Title: The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma

doi: 10.18632/oncotarget.14092

Figure Lengend Snippet: ( A ) C6 glioma cells stably expressing shSpp1 were transfected with various constructs: a control pEGFP, a shRNA resistant, wild type Spp1 (WtSpp1-R) or a shRNA resistant Spp1 lacking a CD44 binding domain (Spp1ΔC-R). Twenty four hours after transfection cells were seeded (8000 cells/ml) under sphere forming conditions (DMEM/F-12 medium with B27, 20 ng/ml bFGF, 20 ng/ml EGF and antibiotics). Reconstitution of Spp1 expression in cells transfected with WtSpp1-R or Spp1ΔC-R was determined by qPCR in respective cultures and related to values obtained for shSpp1 cells. Data are presented as means ± s.d., n = 3. ( B ) Reconstitution of Spp1expression in cells transfected with a control pEGFP, WtSpp1-R or Spp1ΔC-R had no influence cell viability (as determined by MTT metabolism assay 24 h after transfection). ( C ) Only reconstitution of Spp1 expression in glioma cells transfected with a WtSpp1-R restored cell capacity to form spheres. Data are presented as means ± s.d., P values were considered significant when *P ≤ 0.05 and * *P ≤ 0.01, n = 3. ( D ) Representative images show the reduced formation of spheres derived from the shSpp1 glioma cells transfected with pEGFP 7 days after seeding. Overexpression of the construct coding for a wild type (WtSpp1-R) restored sphere forming capacity of glioma cells; overexpression of Spp1ΔC-R did not restore sphere forming capacity.

Article Snippet: CD44 was detected by Western blotting with polyclonal Sheep IgG anti-human CD44 (#AF3660, R&D System, Minneapolis, MN, USA, 1:500) followed by a rabbit Anti-Sheep IgG-HRP conjugate (#AP147P, Merck Millipore KGaA, Darmstadt, Germany, 1:5000).

Techniques: Stable Transfection, Expressing, Transfection, Construct, Control, shRNA, Binding Assay, Derivative Assay, Over Expression

Journal: Oncotarget

Article Title: The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma

doi: 10.18632/oncotarget.14092

Figure Lengend Snippet: Sequences of qPCR primers

Article Snippet: CD44 was detected by Western blotting with polyclonal Sheep IgG anti-human CD44 (#AF3660, R&D System, Minneapolis, MN, USA, 1:500) followed by a rabbit Anti-Sheep IgG-HRP conjugate (#AP147P, Merck Millipore KGaA, Darmstadt, Germany, 1:5000).

Techniques: