Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 8

1.
Figure 1.

Figure 1. From: Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type-1 Expression in Human Pleural Mesothelial Cells.

Expression of plasminogen activator inhibitor type–1 (PAI-1) by MeT5A human pleural mesothelial and MS-1 mesothelioma cells. Total proteins from conditioned media (CM) and cell lysates (CL) of MeT5A and MS-1 cells treated with PBS, TNF-α (10 ng/ml), or TGF-β (2 ng/ml) for 24 hours were subjected to Western blotting using an anti–PAI-1 antibody. The corresponding blots containing proteins from the CL were stripped and reprobed with β-actin monoclonal antibody for assessment of equal loading.

Sreerama Shetty, et al. Am J Respir Cell Mol Biol. 2010 September;43(3):358-367.
2.
Figure 4.

Figure 4. From: Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type-1 Expression in Human Pleural Mesothelial Cells.

Identification of the PAI-1 mRNABp binding sequences of the PAI-1 3′UTR mRNA. (A) Deletion map indicating the PAI-1 mRNABp binding site represented in the PAI-1 mRNA 3′UTR. Positive interactions between the PAI-1 mRNABp and the specific deletion transcript are indicated as (+) or open bars; negative (−) interactions are indicated as solid bars. (B) Met5A cell lysates were incubated with 32P-labeled full-length 3′UTR or 3′UTR deletion transcripts. Transcript–protein complex formation was analyzed by UV cross-linking assay. The arrow indicates the transcript–protein complex. Fp = 32P-labeled PAI-1 mRNA 3′UTR probe incubated with buffer alone.

Sreerama Shetty, et al. Am J Respir Cell Mol Biol. 2010 September;43(3):358-367.
3.
Figure 5.

Figure 5. From: Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type-1 Expression in Human Pleural Mesothelial Cells.

Determination of the destabilization function of the PAI-1 mRNABp binding sequence of the 3′UTR of PAI-1 mRNA. (A) Structure of β-globin–PAI-1 3′UTR chimeric mRNA. The 33-nt (1,690–1,722) PAI-1 3′UTR mRNABp binding sequence and a control non–PAI-1 mRNABp binding sequence of similar size from the coding region were inserted into the 3′UTR of β-globin cDNA, after which the chimeric cDNA was subcloned into pcDNA 3.1. (B) Met5A cells transfected with the chimeric β-globin–PAI-1 3′UTR gene containing the 33 nt PAI-1 mRNABp binding (positive) or nonbinding control (negative) sequence of PAI-1 in pcDNA 3.1. Total RNA was isolated at different time intervals after treatment with DRB as described in the legend to Figure 2, and the level of chimeric mRNA was analyzed by RT-PCR using β-globin forward and PAI-1 reverse primers.

Sreerama Shetty, et al. Am J Respir Cell Mol Biol. 2010 September;43(3):358-367.
4.
Figure 7.

Figure 7. From: Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type-1 Expression in Human Pleural Mesothelial Cells.

Binding of 6-phosphogluconate dehydrogenase (6-PGD) to the 3′UTR PAI-1 mRNA. (A) Porcine citrate (CS) and yeast-derived 6-PGD (10 μg) were incubated with the 32P-labeled PAI-1 mRNA 3′UTR in the presence of tRNA. The reaction mixtures were later digested with RNase T1 and heparin as described in Materials and Methods to avoid nonspecific interaction. After heparin digestion, the reaction mixtures were UV irradiated at 4°C. The immobilized RNA–protein complexes were separated by SDS-PAGE, dried, and autoradiographed. Fp = free probe. (B) Specificity of the yeast 6-PGD–PAI-1 mRNA 3′UTR interaction. Yeast-derived 6-PGD was incubated with a molar excess of unlabeled transcript or homopoly (A), poly (U), poly (G), poly (C) ribonucleotides, or proteinase K or SDS before exposure to 32P-labeled transcript. The reaction mixtures were later digested with RNase T1 and heparin and subjected to UV cross-linking assay as described in (A).

Sreerama Shetty, et al. Am J Respir Cell Mol Biol. 2010 September;43(3):358-367.
5.
Figure 6.

Figure 6. From: Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type-1 Expression in Human Pleural Mesothelial Cells.

Effect of PAI-1 mRNABp binding sequence overexpression on PAI-1. (A) Stable MeT5A cells overexpressing chimeric β-globin–PAI-1 mRNA containing the 33-nt PAI-1 mRNABp binding PAI-1 3′UTR sequence or nonbinding 33-nt CDR sequence were grown to confluence. The cells were switched to serum-free media overnight, and the CM was analyzed for PAI-1 expression by Western blotting using anti–PAI-1 antibody. (B) Total RNA isolated from MeT5A cells overexpressing the PAI-1 mRNABp binding or control sequence was analyzed for PAI-1 mRNA expression by RT-PCR using 32P-labeled deoxycytidine triphosphate (dCTP) in the reaction mixture as described in Figure 2. RNA from the same samples was later analyzed for β-actin mRNA. (C) The MeT5A cells expressing the PAI-1 mRNABp binding or nonbinding control PAI-1 mRNA sequences were treated with DRB to inhibit ongoing transcription and the level of endogenous PAI-1 mRNA was determined by RT-PCR of total RNA collected at different time points. PAI−1 mRNA levels were analyzed by RT-PCR using 32P-labeled dCTP in the reaction mixture. The same sample was analyzed for β-actin mRNA.

Sreerama Shetty, et al. Am J Respir Cell Mol Biol. 2010 September;43(3):358-367.
6.
Figure 2.

Figure 2. From: Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type-1 Expression in Human Pleural Mesothelial Cells.

Effect of TGF-β, phorbol myristate acetate (PMA), and cycloheximide on PAI-1 mRNA expression in MeT5A cells. (A) MeT5A cells treated with PMA (150 ng/ml), TGF-β (2 ng/ml), or cycloheximide (10 μg/ml) for 6 hours. Total RNA was isolated, and PAI-1 mRNA was analyzed by RT-PCR using 32P-labeled deoxycytidine triphosphate in the reaction mixture. The 32P-labled PCR products were separated on urea/polyacrylamide gel electrophoresis, dried, and autoradiographed. (B) MeT5A cells were treated with PBS, PMA, TGF-β, or cycloheximide for 6 hours to induce maximum PAI-1 mRNA. The cells were later treated with 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole (DRB) to inhibit ongoing transcription, and the level of PAI-1 mRNA was determined by RT-PCR of total RNA collected at the selected time points. This data are representative of three repetitions. (C) Graphic depiction of the data illustrated in (B) showing the percentage of maximal (baseline) mRNA for each treatment condition plotted as a function of time.

Sreerama Shetty, et al. Am J Respir Cell Mol Biol. 2010 September;43(3):358-367.
7.
Figure 3.

Figure 3. From: Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type-1 Expression in Human Pleural Mesothelial Cells.

Effect of TGF-β on the PAI-1 mRNABp–PAI-1 mRNA binding interaction. (A) MeT5A cells were treated with PBS or TNF-α (10 ng/ml) or TGF-β (2 ng/ml) for 24 hours at 37°C in serum-free RPMI media. The cytosolic extracts were prepared and separately incubated with 32P-labeled PAI-1 mRNA CDR or 3′UTR in the presence of tRNA. The reaction mixtures were later digested with RNase T1 and heparin as described in Materials and Methods to avoid nonspecific interaction. After heparin digestion, the reaction mixtures were separated on native polyacrylamide gel (4%), dried, and autoradiographed. Fp = free probe. (B) Specificity of the PAI-1 mRNABp–PAI-1 mRNA 3′UTR interaction. Cytosolic extracts of MeT5A cells were incubated with varying amounts of unlabeled transcript before exposure to 32P-labeled transcript. The reaction mixtures were later digested with RNase T1 and heparin and UV irradiated at 4°C. The immobilized RNA–protein complexes were separated on a SDS-PAGE, dried, and autoradiographed. (C) Cytosolic extracts from MeT5A, M33K, M9K, and MS-1 cells were subjected to PAI-1 mRNA 3′UTR binding analyses as described in (A) in the presence of tRNA. After RNase T1 and heparin digestion, the reaction mixtures were subjected to UV cross-linking and autoradiography. The panels are representative of two independent repetitions.

Sreerama Shetty, et al. Am J Respir Cell Mol Biol. 2010 September;43(3):358-367.
8.
Figure 8.

Figure 8. From: Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type-1 Expression in Human Pleural Mesothelial Cells.

Binding of recombinant 6-PGD (r6-PGD) to the 3′UTR PAI-1 mRNA. (A) r6-PGD was expressed in H157 cells. Total lysates from H157 cells transfected with empty pcDNA 3.1 vector (Vc) or 6-PGD cDNA were analyzed for expression of V5 fusion epitope by Western blotting using anti-V5 antibody. (B) Immunoprecipitation of PAI-1 mRNA with r6-PGD. Cell lysates from stable H157 cells transfected with 6-PGD cDNA were sequentially immunoprecipitated with nonspecific mouse IgG and anti-V5 antibodies. Total RNA was isolated from the immune complex, and PAI-1 mRNA was analyzed by RT-PCR in the presence of 32P-labeled dCTP. The PCR reaction containing a PAI-1 cDNA template was used as a positive (+Ve) control, and the reaction lacking template was used as negative (−Ve) control. (C) Purification of r6-PGD. Total lysates from stable H157 cells were passed through a nickel column, after which flow through (FT), wash (W1), and eluates (E1 and E2) were (5 μg) subjected to UV cross-linking (PAI-1 mRNA PGD binding) as described previously to assess the PAI-1 mRNA binding activities of the purified fractions. The same fractions were subjected immunoblotting using anti–6-PGD and anti-V5 antibodies respectively. Specificity of the r6-PGD–PAI-1 mRNA 3′UTR interaction. r6-PGD was incubated with 0- to 400-fold molar excess of unlabeled transcript corresponding to the unlabeled full-length PAI-1 mRNA 3′UTR (D) or 33-nt 6-PGD binding sequence (E) before exposure to 32P-labeled transcript corresponding to the 33-nt PAI-1 mRNABp binding sequence. The reaction mixtures were later digested with RNase T1 and heparin and subjected to UV cross-linking assay as described in Figure 7A. (F) Demonstration of the 6-PGD interaction with PAI-1 mRNA in Met5A cells. Cytosolic extracts (100 μg) were incubated with the 32P-labeled 33-nt 6-PGD binding sequence of PAI-1 mRNA 3′UTR in the presence of tRNA. The reaction mixtures were later digested with RNase T1 and heparin to avoid nonspecific interaction. After UV irradiation of the reaction mixtures at 4°C, the immobilized RNA–protein complexes were immunoprecipitated with protein AG agarose alone (none) or with protein AG agarose conjugated with anti–6-PGD polyclonal antibody (6-PGD) or rabbit IgG (r-IgG). Agarose beads were washed three times with lysis buffer and separated by SDS-PAGE, dried, and autoradiographed. Fp = free probe. (Gi) Stable Met5A cells expressing empty vector (Vc) or 6-PGD cDNA in pcDNA3.1 were switched to serum-free RPMI media overnight. The conditioned media was then analyzed for PAI-1 expression by Western blotting. (Gii) Stable Met5A cells expressing vector or 6-PGD cDNA as described in Figure 8Gi were treated with PBS or TGF-β for 24 hours. The conditioned media was analyzed for PAI-1, and the cell lysates were tested for V5-epitope and β-actin by Western blotting. (Hi) Primary rabbit mesothelial cells cultured in 60-mm dishes were transduced with empty pacAd5CMV vector (Vc) or the same vector carrying 6-PGD cDNA. After 48 hours, the conditioned media were analyzed for PAI-1 expression by Western blotting. (Hii) Primary rabbit mesothelial cells transduced with adenovirus carrying empty vector or 6-PGD cDNA as described in (Hi) were treated with PBS or TGF-β and the conditioned media was analyzed for PAI-1 by Western blotting.

Sreerama Shetty, et al. Am J Respir Cell Mol Biol. 2010 September;43(3):358-367.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk