Journal: The Journal of biological chemistry

Article Title: SPARC regulates the expression of collagen type I and transforming growth factor-beta1 in mesangial cells.

doi: 10.1074/jbc.274.45.32145

Figure Lengend Snippet: FIG. 5. Reduced expression of TGF-b1 mRNA and protein by SPARC-null mesangial cells. A, 1 mg of total RNA extracted from wild-type and SPARC-null cells was reverse-transcribed and amplified in the presence of specific primers for TGF-b1 and for b-tubulin; the transcription products were subjected to agarose gel electrophoresis. Lane 1, wild-type cells; lane 2, SPARC-null cells. B, analysis of condi- tioned media derived from wild-type and SPARC-null cells by an ELISA specific for TGF-b1. The volumes of the respective media analyzed were normalized according to cell number, and levels of TGF-b1 in the growth medium alone are also shown. Results are averaged from five independent experiments.

Article Snippet: Anti-TGF-b1 Antibody Blocking Experiments—To determine whether the effects of exogenous SPARC were exerted through a TGFb1-dependent pathway, we cultured cells as described above and treated them with or without the following: (i) anti-TGF-b1-blocking antibodies (polyclonal goat anti-human IgG, R & D Systems, Inc., Minneapolis, MN) at a final concentration of 30 mg/ml; (ii) rhSPARC (30 mg/ml, 0.9 mM); (iii) rhTGF-b1 (1, 5, and 10 ng/ml); and (iv) either rhSPARC or rhTGF-b1 together with anti-TGF-b1-blocking antibodies for 0–6 h. As a control we used an irrelevant polyclonal goat anti-rabbit IgG (Vector).

Techniques: Expressing, Reverse Transcription, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of biological chemistry

Article Title: SPARC regulates the expression of collagen type I and transforming growth factor-beta1 in mesangial cells.

doi: 10.1074/jbc.274.45.32145

Figure Lengend Snippet: FIG. 6. Induction of TGF-b1 mRNA in mesangial cells by SPARC. Wild-type and SPARC-null cells were stimulated for 6 h with 0.9 mM rhSPARC. Total RNA was prepared and subjected to RT-PCR analysis for TGF-b1 mRNA; b-tubulin mRNA was used as an internal control. Lanes 1 and 3, unstimulated cells; lanes 2 and 4, cells stimu- lated with rhSPARC.

Article Snippet: Anti-TGF-b1 Antibody Blocking Experiments—To determine whether the effects of exogenous SPARC were exerted through a TGFb1-dependent pathway, we cultured cells as described above and treated them with or without the following: (i) anti-TGF-b1-blocking antibodies (polyclonal goat anti-human IgG, R & D Systems, Inc., Minneapolis, MN) at a final concentration of 30 mg/ml; (ii) rhSPARC (30 mg/ml, 0.9 mM); (iii) rhTGF-b1 (1, 5, and 10 ng/ml); and (iv) either rhSPARC or rhTGF-b1 together with anti-TGF-b1-blocking antibodies for 0–6 h. As a control we used an irrelevant polyclonal goat anti-rabbit IgG (Vector).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control

Journal: The Journal of biological chemistry

Article Title: SPARC regulates the expression of collagen type I and transforming growth factor-beta1 in mesangial cells.

doi: 10.1074/jbc.274.45.32145

Figure Lengend Snippet: FIG. 7. Anti-TGF-b1-blocking antibodies reverse the stimula- tory effect of exogenous SPARC on the expression of collagen type I by mesangial cells. Wild-type and SPARC-null cells were stimulated for 0–6 h with rhTGF-b1 (1, 5, and 10 ng/ml) or 0.9 mM rhSPARC in the presence or absence of anti-TGF-b1 blocking antibod- ies. Total RNA was prepared and subjected to RT-PCR. A, RT-PCR analysis of the expression of collagen type I and TGF-b1 after stimula- tion with rhSPARC (0.9 mM) (1) at different time points (0 to 6 h); the induction of collagen type I and TGF-b1 was compared with that of unstimulated cells (2); rpS6 was used as an internal control. Lanes 1–5, wild-type cells; lanes 6–10, SPARC-null cells. B, RT-PCR analysis of the expression of collagen type I after stimulation with different concentra- tions of rhTGF-b1; rpS6 was used as an internal control. Lane C denotes buffer control. Lanes 1–4, wild-type cells; lanes 5–8, SPARC-null cells. C, RT-PCR analysis of collagen type I expression after stimulation with rhTGF-b1 (5 ng/ml), rhSPARC (0.9 mM), and/or anti-TGF-b1 blocking antibodies (30 mg/ml); rpS6 was used as an internal control. Lanes 1–6, wild-type cells; lanes 7–10, SPARC-null cells; lanes 1 and 7, unstimu- lated controls; lanes 2, 5, 8, and 11, stimulation with rhTGF-b1; lanes 3, 6, 9, and 12, stimulation with rhSPARC; lanes 4 and 10, incubation with anti-TGF-b1 blocking antibodies alone; lanes 5 and 11, incubation with anti-TGF-b1-blocking antibodies and rhTGF-b1; lanes 6 and 12, incu- bation with anti-TGF-b1 blocking antibodies and rhSPARC.

Article Snippet: Anti-TGF-b1 Antibody Blocking Experiments—To determine whether the effects of exogenous SPARC were exerted through a TGFb1-dependent pathway, we cultured cells as described above and treated them with or without the following: (i) anti-TGF-b1-blocking antibodies (polyclonal goat anti-human IgG, R & D Systems, Inc., Minneapolis, MN) at a final concentration of 30 mg/ml; (ii) rhSPARC (30 mg/ml, 0.9 mM); (iii) rhTGF-b1 (1, 5, and 10 ng/ml); and (iv) either rhSPARC or rhTGF-b1 together with anti-TGF-b1-blocking antibodies for 0–6 h. As a control we used an irrelevant polyclonal goat anti-rabbit IgG (Vector).

Techniques: Blocking Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Incubation

Journal: Human reproduction (Oxford, England)

Article Title: Altered β1,6-GlcNAc and bisecting GlcNAc-branched N-glycan on integrin β1 are associated with early spontaneous miscarriage in humans.

doi: 10.1093/humrep/dev153

Figure Lengend Snippet: Figure 3 Analysis of b1,6-GlcNAc branching on total human chorionic villous tissue proteins from 6 to 9 weeks (wk) of gestation. (A) Phaseolus vulgaris leucoagglutinin (PHA-L) lectin blotting, demonstrating that the molecular sizes of the major glycoproteins were distributed from ≏70 to 170 kDa. Glyco- proteins from 9 weeks of gestation were more strongly stained with PHA-L than the other studied weeks of gestation (n ¼ 40) during early normal preg- nancies. The numbers on the left indicate the molecular weights of the standards. (B) Lectin blotting using PHA-L revealed decreased b1,6-GlcNAc branching in the ESM group (n ¼ 40) compared with the normal (N) group (n ¼ 60) at each week of gestation. (C) PHA-L lectin fluorescence showed that b1,6-GlcNAc N-glycans were mainly located outside of the STB and CTB layers in normal villi, whereas they were rarely expressed in the ESM villi. Representative images of the three independent experiments are presented. The pictures in the fourth line and eighth line are the higher magnification images of the white boxes in the third line and seventh line. The negative control showed no background staining. Red colour represents the expression of b1,6-GlcNAc branching. The blue colour represents nuclear staining with DAPI. The white arrows indicate CTB; the green arrows indicate STB. Mag- nification of 400×, scale bar: 50 mM.

Article Snippet: For lectin staining, the tissues were blocked with 1× Carbo Free Blocking Solution (Vector Laboratories,Burlingame,USA)andthen incubatedwithbiotinylatedPhaseolusvulgariserythroagglutinin (PHA-E, which binds to bis-GlcNAc structures) or biotinylated P. vulgaris leucoagglutinin (PHA-L, which recognizes b1,6-GlcNAc-branched structures) lectins (Vector Laboratories) at a 1:300 dilution in PBS overnight at 48C.

Techniques: Staining, Negative Control, Expressing

Journal: Human reproduction (Oxford, England)

Article Title: Altered β1,6-GlcNAc and bisecting GlcNAc-branched N-glycan on integrin β1 are associated with early spontaneous miscarriage in humans.

doi: 10.1093/humrep/dev153

Figure Lengend Snippet: Figure 4 Analysis of bis-GlcNAc branching on total human chorionic villous tissue proteins from 6 to 9 weeks (wk) of gestation. (A) Phaseolus vulgaris erythroagglutinin (PHA-E) lectin blotting showed that the molecular sizes of the major glycoproteins were distributed from ≏80–180 kDa. Glycoproteins from 6 weeks of gestation were more strongly stained with PHA-E than other studied weeks of gestation in the normal group. (B) PHA-E lectin blotting revealed that therewasan increased expressionof bis-GlcNAc structures in the ESM group (n ¼ 40) compared with the normal (N) group (n ¼ 60) ateach weekofgestation.(C)PHA-Electinfluorescencerevealedthatthebis-GlcNAcN-glycanswereprimarilylocatedoutsideoftheSTBsinnormalvilli,whereas they were expressed much more abundantly outside of both the STBs and CTBs in the ESM group at each week of gestation. The pictures in the fourth line andeighthlinearethehighermagnificationimagesofthewhiteboxesinthethirdlineandseventhline.ThebluecolourrepresentsnuclearstainingwithDAPI. Redcolour represents bis-GlcNAc branching expression. Thewhite arrows indicate CTB; the green arrows indicate STB. Magnification of 400×, scale bar: 50 mM. N, normal group; ESM, early spontaneous miscarriage group; wk, week of gestation.

Article Snippet: For lectin staining, the tissues were blocked with 1× Carbo Free Blocking Solution (Vector Laboratories,Burlingame,USA)andthen incubatedwithbiotinylatedPhaseolusvulgariserythroagglutinin (PHA-E, which binds to bis-GlcNAc structures) or biotinylated P. vulgaris leucoagglutinin (PHA-L, which recognizes b1,6-GlcNAc-branched structures) lectins (Vector Laboratories) at a 1:300 dilution in PBS overnight at 48C.

Techniques: Staining, Expressing

Journal: Human molecular genetics

Article Title: DelK32-lamin A/C has abnormal location and induces incomplete tissue maturation and severe metabolic defects leading to premature death.

doi: 10.1093/hmg/ddr534

Figure Lengend Snippet: Figure 1. Reduced expression and abnormal maintenance of DK32-lamin A/C in the nucleoplasmic foci in LmnaDK32/DK32 lead to general growth defects and pre- mature death. (A and B) Indirect immunofluorescent confocal micrographs of Lmna+/+ and LmnaDK32/DK32 embryonic muscle sections at E12.5 (A) and E17.5 (B) stained for lamin A/C or lamin B1 (scale bar: 20 mm). (C) Indirect immunofluorescent micrographs of P5 Lmna+/+, Lmna+/DK32 and LmnaDK32/DK32 gastrocnemius sections stained for lamin A/C (scale bar: 20 mm). (D) Representative western blot analysis of Lmna+/+, Lmna+/DK32 and LmnaDK32/DK32 gastrocnemius lysates probed for lamin A/C and lamin B1 proteins relative to actin proteins. (E) Growth curve of Lmna+/+ (n ¼ 32; black diamonds), Lmna+/DK32 (n ¼ 36, grey scares) and LmnaDK32/DK32 (n ¼ 18, white triangles). (F) Representative picture of P12 Lmna+/+ (left; 4.5 cm) and LmnaDK32/DK32 (right; 3.5 cm) mice. (G) Kaplan–Meyer survival curve of Lmna+/+ (n ¼ 22; black line), Lmna+/DK32 (n ¼ 44; large dashed line), LmnaDK32/DK32 (n ¼ 21; dotted line).

Article Snippet: Fluorescent immunostaining was performed using primary antibodies against lamin A/C (N18; sc-6215), lamin B1 (C20; sc-6216), SREBP-1 (K10; sc-367), PPARg (H100; sc-7196) (Santa Cruz), followed by incubation with alexafluorconjugated secondary antibodies (Life Technologies), mounted in Vectashield mounting medium with 4′-6-diamidino-2-phenylindole (Vector Laboratories) and visualized using axiophot fluorescent microscope (Carl Zeiss) for postnatal muscle sections, a confocal microscope (Olympus FV-1000) for liver sections and cultured MEF and an inverted confocal microscope (Leica SP2 AOBS AOTF) for embryonic muscle sections.

Techniques: Expressing, Staining, Western Blot