Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Scheme for the construction of the expression plasmids and the structures of their translation products. cDNAs for rat EGF and human bFGF were amplified by PCR from first-strand cDNAs prepared from the poly(A)+ RNA fraction of rat submaxillary gland and a human osteosarcoma cell line, respectively, using primers tagged with extra 5′ restriction sites. Each amplified cDNA was digested with restriction enzymes and then inserted into the BamHI–EcoRI site of pCHC302, giving pCHC302-EGF and pCHC302-bFGF, which express fusion proteins between GST and collagen-binding EGF (GST-CBEGF) and collagen-binding bFGF (GST-CBFGF), respectively. In the structure of GST-CBEGF (GST-CBFGF), amino acid residues derived from the pGEX-4T-2 plasmid vector are given in the single-letter code, and the thrombin-cleavage site is indicated by an arrow. The numbers in parentheses are the molecular weights of the domains.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Expressing, Amplification, Binding Assay, Derivative Assay, Plasmid Preparation

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Purification profiles of CBEGF and CBFGF on SDS/PAGE. Samples at each purification step for CBEGF (lanes 2–5) and CBFGF (lanes 6–9), and the final preparation of human recombinant bFGF (lane 10) were electrophoretically separated in a SDS/13% polyacrylamide gel under reducing conditions and then stained with Coomassie brilliant blue R-250. Lane 1, molecular weight markers; lane 2, E. coli BL21/pCHC302-EGF crude extract; lanes 3 and 7, eluate from glutathione-Sepharose; lanes 4 and 8, thrombin digest of the eluate from glutathione-Sepharose; lane 5, eluate from Resource Q (purified CBEGF); lane 6, E. coli BL21/pCHC302-bFGF crude extract; lane 9, eluate from heparin-Sepharose (purified CBFGF); lane 10, purified human recombinant bFGF.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Purification, SDS Page, Recombinant, Staining, Molecular Weight

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Dose–response curves for the growth factor activity of rat EGF, CBEGF, human bFGF, and CBFGF. BALB/c 3T3 A31 cells (2 × 104 cells in 2 ml of DMEM-2% calf serum) were inoculated onto 6-well multiwell plates, and 7 h later test samples were added. The cell number was determined with a Coulter counter after 4 days culture. The cell numbers in the absence of test samples and in the presence of 10% calf serum were 29,100 ± 1,600 and 239,600 ± 9,900, respectively. Each point represents the mean value ± SEM for triplicate experiments.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Activity Assay

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Detection of S-phase cells as BrdU incorporation and immunohistochemical staining of CBFGF in subcutaneous tissue of nude mice injected with CBFGF and human bFGF. The animals received an i.p. injection of BrdU (10 mg/100 g body weight) 24 h before death. Immunolocalization was performed on 5-μm paraffin sections by the streptoavidin-biotin-alkaline phosphatase complex technique using anti-BrdU mAbs (a-d) or anti-human bFGF antibodies (e and f) as the primary antibodies. (a, e, and f) 5 days after injection of CBFGF (50 μg). (b) 7 days after injection of CBFGF. (c and d) 5 days and 7 days, respectively, after injection of human bFGF (20 μg). (Bars, 100 μm.)

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: BrdU Incorporation Assay, Immunohistochemical staining, Staining, Injection

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: List of primers for transcript analysis

Article Snippet: Mouse IgG anti-human FGF2 , WB: 1:100 , Santa Cruz , sc-136255.

Techniques:

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: List of antibodies for WB, IF, and flow cytometry analysis

Article Snippet: Mouse IgG anti-human FGF2 , WB: 1:100 , Santa Cruz , sc-136255.

Techniques: Flow Cytometry, Recombinant, Control

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Aging markers in NSCs are affected by late-onset AD. A Scheme illustrating the sample collection of NSCs from two iPSC clones of the same donor. Quantification of B transcript and D protein amounts of the aging markers APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Results are shown as mean ± SEM (* p = 0.05). The bar charts compare three healthy iPSCs (WISCi004-B, WAi001-B, and MLUi009-A) versus four AD iPSCs (MLUi007-H/J and MLUi008-B/F) on 7 d, which were differentiated three times into NSCs ( N = 3 differentiations; healthy: n = 9, AD: n = 12). E Representative image of WB membranes. C Telomere length measured by multiplex QRT-PCR in iPSCs on 0 d and in NSCs on 7 d, shown as the ratio NSCs/iPSCs grouped according to health status ( N = 3 differentiations; healthy: n = 9, AD: n = 12). Results are shown as mean ± SEM (* p = 0.05)

Article Snippet: Mouse IgG anti-human FGF2 , WB: 1:100 , Santa Cruz , sc-136255.

Techniques: Clone Assay, Multiplex Assay, Quantitative RT-PCR

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Detection of marker proteins for cellular aging in NSCs through immunofluorescence (IF). IF analysis showed cellular localization of APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Protein of interest is shown in green versus DNA staining in blue (scale bar, 100 μm)

Article Snippet: Mouse IgG anti-human FGF2 , WB: 1:100 , Santa Cruz , sc-136255.

Techniques: Marker, Immunofluorescence, Staining

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Aging markers in NSCs are affected by APOE genotype. A Transcript and B protein expression analysis of the aging markers APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Results are shown as mean ± SEM (* p = 0.05). The bar charts compare two iPSCs carrying APOE3 (WISCi004-B and MLUi009-A) versus five iPSCs carrying APOE4 (WAi001-B, MLUi007-H/J and MLUi008-B/F) on 7 d, which were differentiated three times into NSCs ( N = 3 differentiations; APOE3: n = 6, APOE4: n = 15). C Telomere length measured by multiplex QRT-PCR in iPSCs on 0 d and in NSCs on 7 d, shown as the ratio NSCs/iPSCs grouped according to APOE status ( N = 3 differentiations; APOE3: n = 6, APOE4: n = 15). Results are shown as mean ± SEM (* p = 0.05). D Manipulation of APOE gene expression by APOE siRNAs and APOE3 plasmids in NSCs generated from reference cell line WISCi004-B on 7 d. No morphological changes are shown by phase contrast imaging up to 4 d after transfection. WB analysis showed the strongest APOE protein repression by APOE siRNAs and strongest APOE induction by APOE3 plasmids on 4 d. Representative images of stained WB membranes are shown (scale bar, 100 μm). Transcript analysis of aging markers in NSCs on 4 d after E APOE inhibition and F APOE induction. Results are shown as mean ± SEM (* p = 0.05). For APOE inhibition, MLUi009-A (healthy matched control, APOE3 carrier) was differentiated into NSCs and transfected with siRNAs on 7 d ( N = 3 differentiations; mock: n = 3, siRNA: n = 3). For APOE induction, the MLUi007-H from (AD, APOE4 carrier) was differentiated into NSCs and transfected with APOE3 plasmids at 7 d ( N = 3 differentiations; mock: n = 3, APOE3 plasmids: n = 3)

Article Snippet: Mouse IgG anti-human FGF2 , WB: 1:100 , Santa Cruz , sc-136255.

Techniques: Expressing, Multiplex Assay, Quantitative RT-PCR, Gene Expression, Generated, Imaging, Transfection, Staining, Inhibition, Control

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Significant mRNA expression changes observed in aging markers

Article Snippet: Mouse IgG anti-human FGF2 , WB: 1:100 , Santa Cruz , sc-136255.

Techniques: Expressing, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Proposed mechanisms for APOE and its impact on aging markers for regulating NSC plasticity. Observed effects on PTEN, ATG7, FGF2, and SIRT1 may reflect the impact of APOE on RTK signaling including FGF2 as key signaling molecule, SIRT1 as transcriptional regulator, and PTEN and ATG7 as target genes. RTKs: receptor tyrosine kinases; JAKs: Janus kinases; AKT: protein kinases B; gray: Golgi apparatus; green arrows: APOE signaling; violet arrows: aging markers signaling via JAKs, RTKs, and Notch

Article Snippet: Mouse IgG anti-human FGF2 , WB: 1:100 , Santa Cruz , sc-136255.

Techniques: