Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: SPARCL1, a Novel Prognostic Predictive Factor for GI Malignancies: a Meta-Analysis.

doi: 10.1159/000485584

Figure Lengend Snippet: Fig. 5. The pooled OR of the association be tween SPARCL1 and GI malig nancies’ lymph nodes metasta sis.

Article Snippet: Goat polyclonal anti-human SPARCL1 IgG antibody (R&D Systems, Minnesota.

Techniques:

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: SPARCL1, a Novel Prognostic Predictive Factor for GI Malignancies: a Meta-Analysis.

doi: 10.1159/000485584

Figure Lengend Snippet: Fig. 6. The pooled OR of the association between SPARCL1 and GI malignancies’ differentiation.

Article Snippet: Goat polyclonal anti-human SPARCL1 IgG antibody (R&D Systems, Minnesota.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

doi: 10.3390/ijms23115874

Figure Lengend Snippet: Effect of SPARC on E-cadherin, vimentin, and ZEB1 expression in prostate cancer cells ( A – C ). Relative expression of SPARC, E-cadherin, and ZEB1 in the prostate cancer cell line DU145 transduced with a lentiviral vector carrying a short hairpin RNA against SPARC (shSPARC) or a scrambled sequence (shScr) ( D ). Relative expression of E-cadherin in the prostate cancer cell line LNCaP treated with different concentrations of SPARC protein or transduced with a lentiviral vector carrying the sequence for SPARC (SPARC_HA) ( A – D ). Relative expression was normalized to pumilio and control cells (first column) using the ΔΔCt method ( E ). Representative Western blot of E-cadherin and vimentin in LNCaP cells treated with 1μg/mL of SPARC protein for up to 72 h ( F , G ). Quantification of the optic density of the Western blot shown in ( F ). Expression of E-cadherin and vimentin was normalized to β-actin ( A – D , F , G ). Data are expressed as mean ± SD ( n = 3). ns = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; Kruskal–Wallis test.

Article Snippet: Recombinant human SPARC protein (Novus Biological, Centennial, CO, USA) was used to stimulate PCa cells at different concentrations.

Techniques: Expressing, Transduction, Plasmid Preparation, shRNA, Sequencing, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

doi: 10.3390/ijms23115874

Figure Lengend Snippet: Effect of SPARC on the expression of integrin subunits and the inhibition of E-cadherin ( A ). Representative Western blot of integrin subunits and SPARC expression in PC3 cells transduced with a lentiviral vector carrying a short hairpin RNA against SPARC (shSPARC) or a scrambled sequence (shScr) (left panel) and in LNCaP cells transduced with a lentiviral vector carrying the SPARC sequence (SPARC_HA) or a control vector (null) (right panel) ( B , C ). Quantification of the optic density of the Western blot shown in ( A ). Expression of integrin subunits and SPARC was normalized to β-actin and control cells ( B , C ). Data are expressed as mean ± SD ( n = 3). ns = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; Mann–Whitney U test.

Article Snippet: Recombinant human SPARC protein (Novus Biological, Centennial, CO, USA) was used to stimulate PCa cells at different concentrations.

Techniques: Expressing, Inhibition, Western Blot, Transduction, Plasmid Preparation, shRNA, Sequencing, Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

doi: 10.3390/ijms23115874

Figure Lengend Snippet: Expression of integrin αvβ3 subunits in prostate cancer cell lines and its effect on the inhibition of E-cadherin ( A , B ). Relative expression of integrin αv (left) and β3 (right) subunits measured by RT-qPCR in the prostate cancer cell lines 22RV1, LNCaP, PC3 and DU145 ( C ). Representative Western blot of integrin αv, β3 subunits and SPARC in different prostate cancer cell lines ( D , E ). Relative expression of E-cadherin ( D ) and vimentin measured by RT-qPCR in the prostate cancer cell LNCaP. LNCaP cells overexpressing SPARC were incubated with different concentrations of RGD peptide ( A , B , D , E ). Relative expression was normalized to pumilio and control cells (first column) using the ΔΔCt method. Data are expressed as mean ± SD ( n = 3). ns = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; Kruskal–Wallis test.

Article Snippet: Recombinant human SPARC protein (Novus Biological, Centennial, CO, USA) was used to stimulate PCa cells at different concentrations.

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Western Blot, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

doi: 10.3390/ijms23115874

Figure Lengend Snippet: Effect of SPARC knockdown on the phosphorylation status of focal adhesion kinase (FAK) and the formation of focal adhesions ( A ). Representative Western blot of FAK, phosphorylated FAK (P-FAK) and SPARC expression in PC3 cells transduced with a lentiviral vector carrying a short hairpin RNA against SPARC (shSPARC) or a scrambled sequence (shScr) ( B ). Quantification of the optic density of the Western blot shown in ( A ). Protein expression was normalized to β-actin and control cells ( C ). Relative expression of RAC1, RHOA, and ILK measured by RT-qPCR in PC3 shScr and PC3 shSPARC cells. Relative expression was normalized to pumilio and control cells (first column) using the ΔΔCt method ( D ). Representative images of immunofluorescence vinculin and cytoskeleton stain with phalloidin in PC3 shScr and PC3 shSPARC cells. Bar = 20 μm ( E ). Quantification of the number of focal adhesions (FA) per cell in both conditions ( F ). Quantification of the area of FA in both conditions ( B , C , E , F ). Data are expressed as mean ± SD ( n = 3). ns = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; Mann–Whitney U test.

Article Snippet: Recombinant human SPARC protein (Novus Biological, Centennial, CO, USA) was used to stimulate PCa cells at different concentrations.

Techniques: Knockdown, Phospho-proteomics, Western Blot, Expressing, Transduction, Plasmid Preparation, shRNA, Sequencing, Control, Quantitative RT-PCR, Immunofluorescence, Staining, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

doi: 10.3390/ijms23115874

Figure Lengend Snippet: Effect of integrin αvβ3 blockade and ZEB1 knockdown on the SPARC-induced E-cadherin downregulation and enhanced migration ( A ). Representative Western blot of ZEB1, E-cadherin and SPARC in LNCaP cells transduced with a lentiviral vector carrying a short hairpin RNA against ZEB1 (shZEB1) or a scrambled sequence (shScr) ( B ). Quantification of the optic density of the Western blot shown in ( A ). Protein expression normalized to β-actin and control cells (shSCR) ( C , D ). Relative expression of E-cadherin measured by RT-qPCR in LNCaP shScramble ( C ) and LNCaP shZEB1 cells stimulated with 1 μg/mL SPARC and 50 μM RGD for 6 h. E-cadherin expression normalized to pumilio and control cells (first column) using the ΔΔCt method ( E , F ). Transwell migration assay of LNCaP shScramble ( E ) and LNCaP shZEB1 ( F ) cells stimulated with 1 μg/mL SPARC and 50 μM RGD for 24 h ( G , H ). Representative images of wound healing assay of LNCaP shScramble ( G ) and LNCaP shZEB1 ( H ) cells stimulated with 1 μg/mL SPARC and 50 μM RGD for 24 h ( I , J ). Quantification of the percentage of the wound area covered after 24 h ( B – F , I , J ). Relative migrations were compared with their own control (basal condition) ( B – F ). Data are expressed as mean ± SD ( n = 3). ns = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; ( B )—Mann–Whitney U test; ( C – F , I , J )—Kruskal–Wallis test.

Article Snippet: Recombinant human SPARC protein (Novus Biological, Centennial, CO, USA) was used to stimulate PCa cells at different concentrations.

Techniques: Knockdown, Migration, Western Blot, Transduction, Plasmid Preparation, shRNA, Sequencing, Expressing, Control, Quantitative RT-PCR, Transwell Migration Assay, Wound Healing Assay, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

doi: 10.3390/ijms23115874

Figure Lengend Snippet: Sequence of oligonucleotides used as primers for the RT-qPCR.

Article Snippet: Recombinant human SPARC protein (Novus Biological, Centennial, CO, USA) was used to stimulate PCa cells at different concentrations.

Techniques: Sequencing, Binding Assay, RNA Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

doi: 10.3390/ijms23115874

Figure Lengend Snippet: Proposed model for the effect of SPARC on E-cadherin expression and migration mediated by integrin α v β 3 and the transcription factor ZEB1 in prostate cancer cells. ( A ) SPARC induces functional changes associated with the epithelial–mesenchymal transition (EMT): increased mesenchymal phenotype and enhanced motility. ( B ) Because SPARC requires the activity of integrin α v β 3 to induce E-cadherin downregulation and cell migration, SPARC effect could be a result of the direct or indirect association of SPARC and the integrin α v β 3 . Through integrin α v β 3 , SPARC could activate the focal adhesion kinase (FAK) and the integrin-linked kinase (ILK). FAK activation by phosphorylation on Y925 promotes focal adhesion turnover, enhancing motility. On the other hand, through FAK, ILK, or other downstream molecules, SPARC could be promoting the expression of the EMT transcription factor ZEB1. The transcription factor ZEB1 inhibits the expression of E-cadherin and induces the expression of mesenchymal markers such as vimentin.

Article Snippet: Recombinant human SPARC protein (Novus Biological, Centennial, CO, USA) was used to stimulate PCa cells at different concentrations.

Techniques: Expressing, Migration, Functional Assay, Activity Assay, Activation Assay, Phospho-proteomics

Journal: Scientific Reports

Article Title: Pancreatic acinar differentiation is guided by differential laminin deposition

doi: 10.1038/s41598-019-39077-6

Figure Lengend Snippet: SPARC and laminin-α1β1γ1, two proteins abundantly found in EPC-CM, control acinar differentiation. ( a ) RT-qPCR analysis of acinar and control genes reported to β-actin in pancreatic explants cultured for 3 days in control medium (Ctrl) and in the presence of SPARC (+SPARC). Addition of SPARC favors acinar differentiation as demonstrated by increased levels of acinar markers ( Cpa and Amy ) and pro-acinar transcription factors ( Rbpjl and Ptf1a ). Expression level of control genes ( Rbpj and E-Cadherin ) are not affected. ( b ) RT-qPCR analysis of acinar and control genes reported to β-actin in pancreatic explants cultured for 3 days in control medium (Ctrl) and in the presence of laminin-α1β1γ1. In the presence of laminin-α1β1γ1, the expression of acinar markers ( Cpa and Amy ) and pro-acinar transcription factors ( Rbpjl and Ptf1a ) is reduced as compared to control explants. Expression level of control genes ( Rbpjl and E-Cadherin ) is not affected. (Mann-Whitney: *p < 0.05). ( c ) Immunofluorescence for the acinar differentiation marker Amylase (green) and the pancreatic epithelium marker (E-Cadherin, red) in explants at 3 days, as indicated. As compared to control explants showing acinar differentiation (Amylase + cells), addition of SPARC stimulates, while laminin-α1β1γ1 decreases the Amylase signal.

Article Snippet: Medium was supplemented with either 1 μg/ml recombinant mouse SPARC (R&D Systems), 2.5 μg/ml purified laminin-α1β1γ1 (gift from T. Sasaki), 3 μM of VEGFR2 inhibitor (SU5416, VWR), or vehicles.

Techniques: Control, Quantitative RT-PCR, Cell Culture, Expressing, MANN-WHITNEY, Immunofluorescence, Marker

Journal: The Journal of Neuroscience

Article Title: Blockade of Astrocytic Calcineurin/NFAT Signaling Helps to Normalize Hippocampal Synaptic Function and Plasticity in a Rat Model of Traumatic Brain Injury

doi: 10.1523/jneurosci.1930-15.2016

Figure Lengend Snippet: Figure7. AAV-Gfa2-VIVITdoesnotalterGFAPorIba1levels,butcausesanincreaseinhevin levels.RepresentativeWesternblots(A,C)andmean SD.GFAPandIba1proteinlevels(B,D) in the contralateral and ipsilateral hippocampus of AAV-treated rats at 7 d after CCI. Note that both glial markers showed a significant increase in the hippocampus of the ipsilateral hemi- sphere, but were not significantly altered by pretreatment with AAV-Gfa2-VIVIT. #p 0.001 ipsilateralversuscontralateral,Fisher’sPLSD.n5–6rats,group.E–H,RepresentativeWest- ern blots (E, G) and mean SD SPARC and hevin protein levels (F, H) in the contralateral and ipsilateralhippocampusofAAV-treatedratsat7dafterCCI.Novirusorinjury-dependenteffects wereobservedforSPARC.Incontrast,hevinwassensitivetobothinjuryandAAVtreatment.In bothAAVgroups,hevinwaselevatedintheipsilateralrelativetothecontralateralhemisphere. OverallhevinlevelsweregreaterintheVIVIT-treatedgroupregardlessofhemisphere,butwere highest in the injured hemisphere. *p 0.05; #p 0.001 ipsilateral versus contralateral, Fisher’s LSD, n 5–6 rats.

Article Snippet: Westerns were performed with the following primary antibodies: GFAP (Cell Signaling Technology, catalog #3670S), Iba1 (Wako, catalog #019-19741), PSD-95 (Cell Signaling Technology, catalog #3450S), synapsin-1 (Cell Signaling Technology, catalog #6710S), GluR1 (Millipore, catalog #AB1504), NR2A (Millipore 07-632), NR2B (Millipore, catalog #05-920), CNA (Millipore, catalog #07-1492), GAPDH (Abcam, catalog #ab9484), hevin (R&D Systems, catalog #MAB2836), and SPARC (R&D Systems, catalog #MAB942).

Techniques:

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 1. Quantification of TSP-1, TSP-2, TNC, and SPARC mRNA expression in human fetal and adult adrenal glands. A, qRT-PCR was performed on RNA isolated from human fetal (14–23 wk, n 7) and adult adrenals (n 4). TSP-1 (open circle), TSP-2 (black triangle), TNC (X), and SPARC (black square) mRNA expression normalized to GUS, an endogenous control. The human adult brain serves as a positive control for expression of the matricellular proteins (values are shown on log scale). B, SPARC mRNA levels in fetal and adult adrenals. *, P 0.005. C, Western blot analysis of SPARC protein in human fetal and adult adrenals. Immunoblot was performed using tissue lysate of a 20-wk human fetal adrenal and human adult adrenal tissue lysate from a commercial source (Pierce). Protein (10 g/lane) was loaded in right two lanes. Human SPARC protein (10 ng) was used as a positive control (PC). Equal loading of protein was confirmed by probing for actin. Densitometric analysis showed 2.8-fold higher expression of SPARC protein in the fetal adrenal than the adult. The blot shown is representative of two experiments with similar results.

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Control, Positive Control, Western Blot

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 4. Effects of forskolin and 8-Br-cAMP on SPARC mRNA levels. FZ cells isolated from human fetal adrenals were exposed to forskolin (F; 1 M) or 8-Br-cAMP (Br; 1 mM) for 48 h. GUS-normalized data (mean SE) are from four independent experiments using different fetal adrenals. *, P 0.05, **, P 0.01 vs. unstimulated, time- matched controls (Co).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Isolation

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 3. A, Dose-dependent effect of ACTH on SPARC and P450c17 mRNA levels. Isolated human fetal adrenal cortical cells (FZ cells) were treated with various concentrations of ACTH for 48 h. Total RNA was extracted and analyzed by qRT-PCR. Constitutively expressed GUS mRNA levels served as normalization controls. Data shown are mean SE of three independent experiments on cells derived from different fetuses. SPARC and P450c17 mRNA levels were increased after exposure to ACTH for 48 h in a dose-dependent manner (P 0.05 and P 0.01, respectively, based on Friedman’s ANOVA). B, Time- dependent effect of ACTH on SPARC mRNA levels. Isolated FZ cells (left panel) or DZ cells (right panel) were treated with ACTH (1 nM) for 24 or 48 h. Each bar represents mean SE of four independent experiments using different fetal adrenals. Black and white bars indicate ACTH-treated and time-matched, unstimulated control cells, respectively. *, P 0.05 vs. control (without ACTH treatment).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Isolation, Quantitative RT-PCR, Derivative Assay, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 2. SPARC protein expression in the human fetal adrenal gland. A, Immunofluorescence of a 22-wk gestation human fetal adrenal gland showing SPARC staining restricted to the FZ. Note the lack of staining in the narrow band corresponding to the DZ. B, Labeling for SPARC (red) and CD56 (green), a DZ cell marker, in a 21-wk human fetal adrenal gland, illustrating the lack of SPARC immunoreactivity in the DZ. C and D, Labeling for SPARC and LDL-R, a FZ cell marker, on an 18-wk human fetal adrenal gland, illustrating FZ-specific lo- calization of SPARC. Note costaining for LDL-R on the membrane (red) and SPARC protein (green) in the cytoplasm in FZ cells. Original magnification, 100 (A and C) and 200 (B and D).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Marker, Membrane

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 6. Zonal expression of mRNAs encoding SPARC, ACTH-R, LDL-R, and P450c17. Outer, DZ and inner, FZ cells in the midges- tation HFA (18–22 wk) were collected using laser capture microdis- section. Total RNA was extracted from cells of the respective zones and analyzed by qRT-PCR as described in Materials and Methods. GUS-normalized data are shown. SPARC, ACTH-R, LDL-R, and P450c17 mRNA levels were observed primarily in the FZ (black bars), compared with those of the DZ (white bars).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 5. Western blot analysis of SPARC protein in cultured human fetal adrenal cortical cells (FZ cells) and conditioned medium. A, The cells were incubated with basal media or in the presence of different concentrations of ACTH (0.001–10 nM) for 48 h. Total cell protein was analyzed by immunoblot. Fifty micrograms of protein were loaded per lane. Data (mean SE) are the densitometric units of SPARC relative to actin from three experiments using different fetal adrenals. The relative ratio of unstimulated cells (control) is arbitrarily presented as 1. SPARC protein levels in cell lysates were increased after expo- sure to ACTH for 48 h in a dose-dependent manner (P 0.05, based on Kruskal-Wallis ANOVA). A representative blot from a 21-wk-old human fetal adrenal is shown above. B, Exposure to 8-Br-cAMP (Br; 1 mM) for 48 h increased abundance of SPARC protein in cell lysates (50 g protein/lane), mimicking the effects of ACTH (Ac; 1 nM). The summary of densitometric evaluation of three independent experi- ments is shown below. Data (mean SE) are the densitometric units of SPARC relative to actin, and the relative ratio of the control (Co; unstimulated cells) is arbitrarily presented as 1. A representative blot from a 19-wk-old human fetal adrenal is shown above. C, Exposure to 8-Br-cAMP (Br; 1 mM) for 48 h increased SPARC secretion into the conditioned medium, mimicking the effects of ACTH (Ac; 1 nM). Total protein (150 g protein) was loaded in each lane, and equal loading was confirmed by the Ponceau S staining. Densitometry is shown for three experiments using different fetal adrenals, with a representa- tive blot from a 22-wk-old human fetal adrenal. The relative ratio of the control (Co; unstimulated cells) is arbitrarily presented as 1. *, P 0.05, **, P 0.01, based on Mann-Whitney U test in comparison with respective control (unstimulated cells).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Western Blot, Cell Culture, Incubation, Control, Staining, MANN-WHITNEY, Comparison