Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A negatively regulates the TGF-β/SMAD signaling pathway. (A) The effect of KDM6A overexpression on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in Huh7 and LM3 cells was (A) determined by western blotting and (B) semi-quantified. The effect of KDM6A knockdown on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in YY-8103 and SNU-398 was (C) determined by western blotting and (D) semi-quantified. ** P<0.01 and *** P<0.001 vs. vector or SCR. KDM6A, lysine demethylase 6A; TGF-β, transforming growth factor-β; p, phosphorylated; PCNA, proliferating cell nuclear antigen; SCR, scrambled; sh, short hairpin RNA; ns, not significant.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Over Expression, Expressing, Western Blot, Plasmid Preparation, shRNA

Journal: Journal of Biological Chemistry

Article Title: Oncogenic Ras Represses Transforming Growth Factor-β/Smad Signaling by Degrading Tumor Suppressor Smad4

doi: 10.1074/jbc.m100069200

Figure Lengend Snippet: FIG. 1. Effect of oncogenic Ras on Smad4 expression and TGF- b-induced Smad complex formation. A, time kinetics of oncogenic Ras expression. Ha-Ras expression was analyzed after addition of IPTG to RIE:iRas cells for 0–72 h. Lysates were collected at different hours and used for immunoblotting using Pan-Ras monoclonal antibodies. B, time kinetics of Smad4 expression. RIE:iRas cells were treated with (1/2)-IPTG for 0–72 h and subjected to immunoblotting to detect the Smad4 expression. The same membrane was probed with anti-b-actin monoclonal antibodies and provided as loading control. C, Smad4 ex- pression in mink, RIE-1, RIE-Ha-Ras, RIE:iRas, and SW 480.7 cells was analyzed. 50 mg of cell lysates were used for immunoblotting with anti-human Smad4 monoclonal antibodies. D, degradation of Smad4 in RIE:iRas cells treated with (2/1)-IPTG. E, time kinetics Smad4 com- plex formation in RIE:iRas cells. RIE:iRas cells were treated with (1/2)-IPTG for 0–72 h and then exposed to TGF-b1 (200 pM) in serum- free media for 30 min. Complex formation was analyzed at each time point by co-immunoprecipitation analysis as mentioned before. The same lysates were used to detect the TGF-b1-induced carboxyl-terminal phosphorylation of Smad2 using rabbit polyclonal antibodies raised against the peptide corresponding to amino acids 457–467 of Smad2. The Smad2/3 level was also determined by immunoblotting using rabbit polyclonal antibodies.

Article Snippet: Beads were washed thoroughly (4 times) with phosphate-buffered saline containing 0.1% Nonidet P-40 and 0.1% SDS and subjected to Western blot analysis using anti-human Smad4 monoclonal antibody (SC 7966, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Bioprocessing, Membrane, Control, Immunoprecipitation, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Oncogenic Ras Represses Transforming Growth Factor-β/Smad Signaling by Degrading Tumor Suppressor Smad4

doi: 10.1074/jbc.m100069200

Figure Lengend Snippet: FIG. 2. Reversible Smad4 expression, TGF-b-mediated com- plex formation. A, Northern blot analysis of Smad4 mRNA. Total RNA was isolated from RIE:iRas cells at different hours after treatment with (1/2)-IPTG by Tri-Reagent, and 20 mg of total RNA was used for detecting the Smad4 mRNA. Cyclophilin A message (1B15) was used as control to determine the integrity of RNA and equality of loading. B, recovery of Smad4 expression; C, Smad4 complex formation upon with- drawal of IPTG. RIE:iRas cells were treated with IPTG for 72 h, sub- cultured, and released from IPTG; culturing of the cells continued up to 96 h. Ha-Ras expression was also analyzed at the same time.

Article Snippet: Beads were washed thoroughly (4 times) with phosphate-buffered saline containing 0.1% Nonidet P-40 and 0.1% SDS and subjected to Western blot analysis using anti-human Smad4 monoclonal antibody (SC 7966, Santa Cruz Biotechnology).

Techniques: Expressing, Northern Blot, Isolation, Control, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Oncogenic Ras Represses Transforming Growth Factor-β/Smad Signaling by Degrading Tumor Suppressor Smad4

doi: 10.1074/jbc.m100069200

Figure Lengend Snippet: FIG. 3. Oncogenic Ras inhibits TGF-b-mediated growth inhibi- tion. A, oncogenic Ras and TGF-b-mediated [3H]thymidine incorpora- tion. Cells, seeded in 24-well plates, were maintained and incubated with and without IPTG for 24–72 h. Then IPTG was withdrawn and cells were cultured for another 72 h at each time point. TGF-b was added for the final 22 h followed by the 2-h incubation with 1 mCi/ml [3H]thymidine. The radioactivity incorporated into the trichloroacetic acid material was counted by a liquid scintillation counter. Incorpora- tion of [3H]thymidine at each time point (1/2IPTG) in the absence of TGF-b was considered as 100% growth. All data shown are means 6 S.D. B, RIE:iRas cells were infected with recombinant adenovirus con- taining either F-Smad4 or b-gal cDNA at the multiplicity of infection of 20 in the presence or absence of IPTG as indicated. After 24 h of infection, cells were washed with DMEM and treated with (1/2) 200 pM TGF-b in (1/2)-IPTG for 22 h followed by a 2-h incubation with 1 mCi/ml [3H]thymidine. The radioactivity incorporated into the trichlo- roacetic acid material was counted by a liquid scintillation counter. Incorporation of [3H]thymidine at each time point (1/2IPTG) in the absence of TGF-b was considered as 100% growth. All data shown are means 6 S.D. C, RIE:iRas cells were infected with recombinant adeno- virus as indicated. After 24 h, cells were washed with DMEM and further incubated for another 24 h. Cells were lysed after 48 h and immunoprecipitated with anti-FLAG antibody, and precipitates were analyzed with anti-Smad4 monoclonal antibodies.

Article Snippet: Beads were washed thoroughly (4 times) with phosphate-buffered saline containing 0.1% Nonidet P-40 and 0.1% SDS and subjected to Western blot analysis using anti-human Smad4 monoclonal antibody (SC 7966, Santa Cruz Biotechnology).

Techniques: Incubation, Cell Culture, Radioactivity, Infection, Recombinant, Virus, Immunoprecipitation, Bioprocessing

Journal: Journal of Biological Chemistry

Article Title: Oncogenic Ras Represses Transforming Growth Factor-β/Smad Signaling by Degrading Tumor Suppressor Smad4

doi: 10.1074/jbc.m100069200

Figure Lengend Snippet: FIG. 4. Oncogenic Ras causes an increased rate of Smad4 deg- radation. RIE:iRas cells were treated without IPTG (A) and with IPTG (B) for 60 h. They were labeled with 150 mCi/ml [35S]methionine (Trans35S-label) for 5 h and then chased in cold methionine-containing medium for the indicated time period. An equal amount of protein (600 mg) from each sample was subjected to immunoprecipitation, resolved by SDS-polyacrylamide gel electrophoresis, and visualized by autora- diography. The intensity of the bands was quantitated by densitometry (alpha-Imager). Steady state level of the Smad4 at identical conditions was determined by immunoblotting. C, the relative levels of Smad4 in the absence or presence of IPTG. This experiment was repeated twice.

Article Snippet: Beads were washed thoroughly (4 times) with phosphate-buffered saline containing 0.1% Nonidet P-40 and 0.1% SDS and subjected to Western blot analysis using anti-human Smad4 monoclonal antibody (SC 7966, Santa Cruz Biotechnology).

Techniques: Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Oncogenic Ras Represses Transforming Growth Factor-β/Smad Signaling by Degrading Tumor Suppressor Smad4

doi: 10.1074/jbc.m100069200

Figure Lengend Snippet: FIG. 5. Restoration of Smad4 in the presence of oncogenic Ras using the inhibitor of ubiquitin proteasome and MEK. A, cells were treated with IPTG along with 5 mM ALLN as indicated. After 72 h cells were treated with TGF-b1 for 30 min. Lysates were analyzed for Smad4 expression and complex formation. The same lysates were also used to evaluate the relative expression of Smad2/3 and TGF-b1-in- duced carboxyl-terminal phosphorylation of Smad2. B, cells were treated with (2/1)-IPTG along with 5 mM lactacystin as indicated. After 72 h cells were lysed and subjected to immunoblot analysis using Smad4 monoclonal antibodies. C, effect of MAP kinase inhibitors on Smad4 expression and complex formation. Cells were treated with PD98059 at 10 mM and SB28059 at 5 mM with (1/2)-IPTG for 72 h. After incubation, cells were washed with phosphate-buffered saline and sub- jected to further incubation with TGF-b1 as indicated for 30 min. Cell lysates were analyzed for Smad4 expression, complex formation, and Ha-Ras expression under similar conditions.

Article Snippet: Beads were washed thoroughly (4 times) with phosphate-buffered saline containing 0.1% Nonidet P-40 and 0.1% SDS and subjected to Western blot analysis using anti-human Smad4 monoclonal antibody (SC 7966, Santa Cruz Biotechnology).

Techniques: Ubiquitin Proteomics, Expressing, Phospho-proteomics, Western Blot, Bioprocessing, Incubation, Saline

Journal: Journal of Biological Chemistry

Article Title: Oncogenic Ras Represses Transforming Growth Factor-β/Smad Signaling by Degrading Tumor Suppressor Smad4

doi: 10.1074/jbc.m100069200

Figure Lengend Snippet: FIG. 6. Oncogenic Ras and TGF-b1-mediated nuclear translo- cation, transcriptional responses. A, nuclear localization of Smad4 after induction of oncogenic Ras. RIE:iRas cells were treated with IPTG for 72 h, then released from IPTG and continued culturing for another 72 h. After treatment, cells were exposed to TGF-b1 for 30 min. Cells were fixed with 4% paraformaldehyde and stained with Smad4 mono- clonal antibodies and CY-3 conjugated goat antimouse IgG. B, upper panel, oncogenic Ras and TGF-b-mediated transcriptional responses. RIE:iRas cells were transiently transfected with p3TP-Lux (0.7 mg), b-galactosidase (0.3 mg), and increasing amounts (0.5 and 1.0 mg) of carboxyl-terminally HA-tagged Smad4 as indicated in the presence or absence of IPTG. Cells were treated with 1/2-TGF-b1 (200 pM) for the final 20 h along with the IPTG in 0.2% serum-containing medium. Cell lysates were analyzed for luciferase activity and were normalized to the b-galactosidase activity. Lower panel, RIE:iRas cells were transiently transfected with HA-Smad4 in the presence or absence of IPTG. After 48 h cells were lysed and immunoprecipitated with anti-HA antibody, and precipitates were analyzed with anti-Smad4 monoclonal antibodies.

Article Snippet: Beads were washed thoroughly (4 times) with phosphate-buffered saline containing 0.1% Nonidet P-40 and 0.1% SDS and subjected to Western blot analysis using anti-human Smad4 monoclonal antibody (SC 7966, Santa Cruz Biotechnology).

Techniques: Staining, Transfection, Luciferase, Activity Assay, Immunoprecipitation, Bioprocessing

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A) PLA utilizes a pair of oligonucleotide-conjugated secondary antibodies that bind corresponding antibodies targeting each interacting protein partner. The proximity of these two protein partners allows circular DNA amplification of the oligonucleotides template upon in situ ligation. Fluorescent probes complementary to this amplicon engender a detectable signal via fluorescence microscopy. Plasma membrane permeabilization was used for intracellular protein interactions. (B and C) PLA indicating protein-protein interactions of PAI-1 and low-density LRP1 on the cell surface (B) or intracellular (C) due to ASC-CM exposure in the presence or absence of the LRP1 inhibitor RAP. Representative micrograph images showing positive signal (red) for PAI-1/LRP1 interactions. DAPI was used to stain nuclei (blue). (D) ChIP-qPCR analysis of LRP1-ICD binding to two promoter regions of the SMAD4 gene in control (Ctrl) cells and CM-exposed cells treated and untreated with RAP (5 μg/mL). Data represent mean ± SD, n = 3 technical replicates; **p < 0.01, ***p < 0.001. (E) qRT-PCR analysis of mRNA expression levels of SMAD4 gene in control cells and CM-exposed cells treated and untreated with Tip (50 μM) or RAP (5 μg/mL). Data represent mean ± SD, n = 3 technical replicates; *p < 0.05. (F) PLA demonstrating protein-protein interactions of SMAD4 and ubiquitin due to ASC-CM exposure in the presence or absence of the following inhibitors: Tip for PAI-1, RAP for LRP1, and protease inhibitor MG132. Micrograph images showing positive PLA signal (green) for SMAD4-ubiquitin interactions. DAPI was used to stain nuclei (blue). (G) Protein expression of LRP1 and SMAD4 by capillary western immunoassays. Endometrial epithelial EME6/7t cells were treated with control media, ASC-CM, and ASC-CM plus Tip, or with pre-treatment with LRP1 antagonist RAP or protease inhibitor MG132. The result shown is a representative of three independent experiments. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: DNA Amplification, In Situ, Ligation, Amplification, Fluorescence, Microscopy, Clinical Proteomics, Membrane, Protein-Protein interactions, Staining, ChIP-qPCR, Binding Assay, Control, Quantitative RT-PCR, Expressing, Ubiquitin Proteomics, Protease Inhibitor, Western Blot

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A) Bar plots showing mRNA expression levels of selected PAI1-regulated JAC genes by qRT-PCR in the two sh SMAD4 knockdown clones, compared to shCtrl in the presence or absence of ASC-CM. (B) ChIP-qPCR analysis of SMAD4 binding to promoter regions of the JAC genes in control (Ctrl) cells and ASC-CM-exposed cells. Data represent mean ± SD, n = 3 technical replicates; *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Clone Assay, ChIP-qPCR, Binding Assay, Control

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A and B) tSNE plots of mass cytometry (CyTOF) analysis visualizing 74,048 epithelial cells, which were divided into clusters (n = 27) based on their proteomic profiles (A). These cells included immortalized EEC line EME6/7t (exposed to ASC-CM ± Tip) and primary endometrial tumors of patients with known BMIs (B). (C) tSNE-based phenographs showing abundance and cellular distributions of JAC proteins. (D) tSNE-based phenographs showing EpCAM and CK8/18 as epithelial cell markers and TGFR-2, SMAD2, and SMAD4 as TGF-β factors. See also and and .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Mass Cytometry

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A–C) Expression profiles of SMAD4 and JAC based on CyTOF were classified into five categories: I, II, III, IV, and V. Violin plots show ordered SMAD4 expression level (low to high) and distribution in the individual subpopulations (A). Expression heatmaps of JAC were aligned according to SMAD4 expression levels (in descending order) in different cellular subpopulations (B). Area of circle denotes relative cell proportion of a subpopulation within each sample and ordered in the aforementioned five categories in cellular subpopulations of EME6/7t lines and primary tumors (C). Tumors were ordered from top to bottom based on increased patients’ BMIs and histology classes. (D) Balloon plots of subpopulation distributions of endometrial tumors in the five categories based on histology and BMI. Circle area indicates the size of each subpopulation. Categories IV and V were associated with relatively high expression levels of SMAD4 and JAC, whereas low levels of SMAD4 and JAC were noted in categories I, II, and III. (E) Violin plots showing the average expression levels of four JAC markers—ADAM15, ROCK1, Cx43, and Gas1—based on patients’ tumor histology: papillary serous and endometrioid. (F) Balloon plots of subpopulation distributions in the five categories stratified by BMI into three groups: normal weight (NW), obesity (OB), and morbid obesity (MOB). (G) Violin plots showing the average expression levels of ADAM15, ROCK1, Cx43, and Gas1, based on three BMI groups: NW, OB, and MOB. Lower JAC expressions corresponded with patients with higher BMIs. ****p < 0.0001. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Expressing

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Ubiquitin Proteomics, Recombinant, Multiplex Assay, Reverse Transcription, SYBR Green Assay, In Situ, shRNA, Software

Journal: The Journal of allergy and clinical immunology

Article Title: Gain-of-Function Variants in SMAD4 Compromise Respiratory Epithelial Function

doi: 10.1016/j.jaci.2024.08.024

Figure Lengend Snippet: A. Representative western blot image showing that SMAD4, SMAD2/3, and SMAD2/3 phosphorylation (p-SMAD2/3) are similar in healthy control (HC#1) and SMAD4-I500Val nasal basal cells after 2 hours of 10 ng/ml TGFβ1 stimulation. B-D. Quantification of SMAD4, SMAD2/3, and p-SMAD2/3 in healthy control (n=3) and Myhre (n=3) nasal basal cells after 2 hours of 10 ng/ml TGFβ1 stimulation. n.s: not significant. E. Transcriptomic analysis (heatmap) revealed that many genes associated with TGF/BMP/SMAD signaling pathways are not differentially expressed in healthy control (n=3) and Myhre (n=3) nasal basal cells ( GSE270668 ).

Article Snippet: The following primary antibodies were utilized: SMAD4 (Most SMAD4 western blotting in this work was performed using the antibody obtained from Origene, TA303589, Rabbit IgG.

Techniques: Mutagenesis, Phospho-proteomics, Gene Expression, Protein-Protein interactions, Western Blot, Control

Journal: The Journal of allergy and clinical immunology

Article Title: Gain-of-Function Variants in SMAD4 Compromise Respiratory Epithelial Function

doi: 10.1016/j.jaci.2024.08.024

Figure Lengend Snippet: A. Schematic of TGFβ/SMAD-luciferase assay in nasal basal cells and differentiated epithelial cells with or without 24 hours of TGFβ1 stimulation. B-C. Quantification of luciferase activity in nasal basal cells (B) and differentiated epithelial cells (C) with or without 24 hours of TGFβ1 stimulation (relative to healthy controls without TGFβ1 stimulation). mean ± SEM, ***P < 0.001. n=3 independent experiments. D. Representative western blot images showing the hypermorph PPIs induced by SMAD4-I500V variant. HEK293T cells were co-expressed with the GST-SMAD4 WT or I500V together with Venus-flag (VF)-tagged SMAD3. GST pull-down (PD) and the whole-cell lysates (WCL) were analyzed by western blotting. E. Bar graph showing the differential SMAD4-SMAD3 PPIs induced by indicated SMAD4 variants. PPI signals were measured by TR-FRET using HEK293 cell lysate co-expressing the His-SMAD4 WT or indicated variants with flag-tagged SMAD3. mean ± SEM, *P < 0.05, n=3 independent experiments.

Article Snippet: The following primary antibodies were utilized: SMAD4 (Most SMAD4 western blotting in this work was performed using the antibody obtained from Origene, TA303589, Rabbit IgG.

Techniques: Mutagenesis, Activity Assay, Luciferase, Western Blot, Variant Assay, Expressing

Journal: Nature genetics

Article Title: Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome.

doi: 10.1038/ng.3512

Figure Lengend Snippet: Figure 4 NUP93, importin 7 and exportin 5 interact with SMAD proteins, and NUP93 mutations from individuals with SRNS abrogate the interaction of NUP93 with SMAD4 and importin 7 in coimmunoprecipitation in HEK293T cells. (a) C-terminally GFP-tagged human NUP93 precipitates endogenous SMAD4. (b) C-terminally GFP-tagged human SMAD4 precipitates endogenous NUP93. (c) Upon co-overexpression, GFP-tagged human NUP93 interacts with Myc-tagged human SMAD4. Mutants (Lys442Asnfs*14, Gly591Val, Tyr629Cys) identified in individuals with SRNS have abrogated SMAD4 interaction. (d) Upon BMP7 stimulation, C-terminally GFP-tagged human NUP93 interacts with phosphorylated, activated SMAD1/5 (p-SMAD1/5). (e) C-terminally GFP-tagged human NUP93 precipitates endogenous importin 7. (f) C-terminally GFP-tagged human SMAD4 precipitates endogenous importin 7. (g) Upon co-overexpression, GFP-tagged human NUP93 interacts with Myc-tagged human importin 7. Mutants (Lys442Asnfs*14, Gly591Val, Tyr629Cys) identified in individuals with SRNS have abrogated importin 7 interaction. (h) GFP-tagged human SMAD4 precipitates endogenous exportin 5. Deletion of the SMAD4 nuclear export signal (NES; residues 142–149) abrogates the interaction with exportin 5. Triangles indicate the predicted molecular weights of precipitated proteins. The coimmunoprecipitation experiments shown in a, b and d–f were confirmed using N-terminally GFP-tagged fusion proteins.

Article Snippet: For immunoblotting, the following primary antibodies were used: mouse antibody to NUP93 (F2), sc-374400, Santa Cruz Biotechnology diluted 1:500; mouse antibody to SMAD4 (B-8), sc-7966, Santa Cruz Biotechnology diluted 1:500; goat antibody to importin 7, NB100-1081, Novus Bioscience diluted 1:1,000; rabbit antibody to NUP205, HPA024574, Atlas Antibodies, Sigma diluted 1:1,000; and rabbit antibody to phosphorylated Smad1/5, 9526, Cell Signaling Technology diluted 1:1,000.

Techniques: Over Expression

Journal: Cell Cycle

Article Title: SQSTM1/p62 regulates the expression of junctional proteins through epithelial-mesenchymal transition factors

doi: 10.4161/15384101.2014.987619

Figure Lengend Snippet: p62 regulates the expression of junctional proteins via the Smad/Snail signaling pathway. (A) Snail mRNA levels were quantified by qRT-PCR in MDCK and MDCKp62 cells treated with TGFβ from 0 to 96 h. (B) MDCK, MDCKp62 cells, and MDCK cells treated with p62 siRNA were subjected to TGFβ treatment. Expression of Snail and actin were revealed by protein gel blotting. (C) MDCK and MDCKp62 cells were treated or not with Smad4 siRNA and subjected to TGFβ treatment. Expression of Smad4, Snail, and actin were revealed by western blotting. (D) MDCK and MDCKp62 cells were treated with TGFβ for 72 h. Expression of Smad4 and PCNA were revealed by protein gel blotting of nuclear extracts. (E) MDCK, MDCKp62 cells or siRNA p62-treated MDCK cells were transiently transfected with a Smad reporter construct together with a Renilla luciferase expression vector and treated with TGFβ for the indicated times.

Article Snippet: Antibodies Antibodies directed against HA and Smad4 (Santa Cruz), Smad2/3 (BD Transduction), E-cadherin (BD Transduction and clone rr1, Developmental Studies Hybridoma Bank, University of Iowa), Snail, PCNA, (Cell Signaling), Twist (Aviva Systems Biology), actin (clone C4, Millipore), p62 (Abnova), occludin and claudin1/3 (Invitrogen), Flag (Sigma), GFP (Roche), Myc-tag, LC3B (Sigma), histidine (Cell Signaling), Ubiquitin (Pierce), horseradish peroxidase-linked secondary antibodies (Jackson Immunology and Rockland for TrueBlot ULTRA antibodies) were used for protein gel blot analyses.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Construct, Luciferase, Plasmid Preparation

Journal: Cell Cycle

Article Title: SQSTM1/p62 regulates the expression of junctional proteins through epithelial-mesenchymal transition factors

doi: 10.4161/15384101.2014.987619

Figure Lengend Snippet: The UBA domain of p62 is required to stabilize Smad4. (A) MDCK, MDCKp62 cells, and MDCK cells treated with p62 siRNA were subjected to TGFβ treatment. p62, Smad4, and actin were detected by immunoblotting (LE: low exposition, HE: high exposition). (B) Immunoblot analysis of Smad4 and actin in control and MDCK cells that overexpress wild-type p62, p62ΔUBA or p62 4XNLS; cells were treated with TGFβ for 48 h and cycloheximide (CHX) for the last 6 or 8 h. (C) MDCK, NMuMG, and NBT-II cells were treated or not with TGFβ for 48 h and 24 h respectively. Immunoprecipitation (IP) was performed using an anti-p62 antibody or a monoclonal control IgG. Smad4, p62 and actin were revealed by immunoblotting.

Article Snippet: Antibodies Antibodies directed against HA and Smad4 (Santa Cruz), Smad2/3 (BD Transduction), E-cadherin (BD Transduction and clone rr1, Developmental Studies Hybridoma Bank, University of Iowa), Snail, PCNA, (Cell Signaling), Twist (Aviva Systems Biology), actin (clone C4, Millipore), p62 (Abnova), occludin and claudin1/3 (Invitrogen), Flag (Sigma), GFP (Roche), Myc-tag, LC3B (Sigma), histidine (Cell Signaling), Ubiquitin (Pierce), horseradish peroxidase-linked secondary antibodies (Jackson Immunology and Rockland for TrueBlot ULTRA antibodies) were used for protein gel blot analyses.

Techniques: Western Blot, Control, Immunoprecipitation

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

doi: 10.1002/ar.23610

Figure Lengend Snippet: Figure 1. Immunohistochemical staining of various proteins in colon cancer cells. (A) Smad4 expressed in

Article Snippet: Formalin-fixed paraffin sections (4 μm thick) were stained with anti-Smad4 monoclonal antibody (1:150 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-A polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-C polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-CD34 antibody (1:50 dilution; Abcam, MA, USA) or anti-D2-40 (1:30 dilution; Abcam, MA, USA) for immunohistochemistry in accordance with the instructions, and the poly-horseradish peroxidase system and 3,3'-diaminobenzidine (Zhongshan Biotechnology Inc., Beijing, China) were used.

Techniques: Immunohistochemical staining, Staining

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

doi: 10.1002/ar.23610

Figure Lengend Snippet: Figure 3. Effects of pcDNA-Smad4 on Smad4 expression in colon cancer cells. For selection of a colon cancer

Article Snippet: Formalin-fixed paraffin sections (4 μm thick) were stained with anti-Smad4 monoclonal antibody (1:150 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-A polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-C polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-CD34 antibody (1:50 dilution; Abcam, MA, USA) or anti-D2-40 (1:30 dilution; Abcam, MA, USA) for immunohistochemistry in accordance with the instructions, and the poly-horseradish peroxidase system and 3,3'-diaminobenzidine (Zhongshan Biotechnology Inc., Beijing, China) were used.

Techniques: Expressing, Selection

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

doi: 10.1002/ar.23610

Figure Lengend Snippet: Figure 4. Effects of Smad4 on the biological behavior of HCT-116 cells. (A) MTT assays evaluated the effects of Smad4 on HCT-116 cell proliferation. Smad4 overexpression had no significant effect on proliferation. (B)

Article Snippet: Formalin-fixed paraffin sections (4 μm thick) were stained with anti-Smad4 monoclonal antibody (1:150 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-A polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-C polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-CD34 antibody (1:50 dilution; Abcam, MA, USA) or anti-D2-40 (1:30 dilution; Abcam, MA, USA) for immunohistochemistry in accordance with the instructions, and the poly-horseradish peroxidase system and 3,3'-diaminobenzidine (Zhongshan Biotechnology Inc., Beijing, China) were used.

Techniques: Over Expression

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

doi: 10.1002/ar.23610

Figure Lengend Snippet: Figure 5. Smad4 affects the expressions of VEGF-A and -C in HCT-116 cells. VEGF-A (A) and VEGF-C (B) mRNA expressions were detected by qRT-PCR. VEGF-A (C) and VEGF-C (D) protein levels were detected by

Article Snippet: Formalin-fixed paraffin sections (4 μm thick) were stained with anti-Smad4 monoclonal antibody (1:150 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-A polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-C polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-CD34 antibody (1:50 dilution; Abcam, MA, USA) or anti-D2-40 (1:30 dilution; Abcam, MA, USA) for immunohistochemistry in accordance with the instructions, and the poly-horseradish peroxidase system and 3,3'-diaminobenzidine (Zhongshan Biotechnology Inc., Beijing, China) were used.

Techniques: Quantitative RT-PCR

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

doi: 10.1002/ar.23610

Figure Lengend Snippet: Figure 6. Effects of Smad4 on TGF-β1 and TLP expression and Smad2 and Smad3 phosphorylation. Both

Article Snippet: Formalin-fixed paraffin sections (4 μm thick) were stained with anti-Smad4 monoclonal antibody (1:150 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-A polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-C polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-CD34 antibody (1:50 dilution; Abcam, MA, USA) or anti-D2-40 (1:30 dilution; Abcam, MA, USA) for immunohistochemistry in accordance with the instructions, and the poly-horseradish peroxidase system and 3,3'-diaminobenzidine (Zhongshan Biotechnology Inc., Beijing, China) were used.

Techniques: Expressing, Phospho-proteomics

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

doi: 10.1002/ar.23610

Figure Lengend Snippet: Figure 7. Effects of Smad4 on the colon cancer xenografts in nude mice. (A) Smad4 overexpression inhibited

Article Snippet: Formalin-fixed paraffin sections (4 μm thick) were stained with anti-Smad4 monoclonal antibody (1:150 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-A polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-VEGF-C polyclonal antibody (1:200 dilution; Zhongshan Biotechnology Inc., Beijing, China), anti-CD34 antibody (1:50 dilution; Abcam, MA, USA) or anti-D2-40 (1:30 dilution; Abcam, MA, USA) for immunohistochemistry in accordance with the instructions, and the poly-horseradish peroxidase system and 3,3'-diaminobenzidine (Zhongshan Biotechnology Inc., Beijing, China) were used.

Techniques: Over Expression

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Ligustrazine Attenuates Liver Fibrosis by Targeting miR-145 Mediated Transforming Growth Factor- β /Smad Signaling in an Animal Model of Biliary Atresia.

doi: 10.1124/jpet.121.001020

Figure Lengend Snippet: Fig. 2. Ligustrazine inhibited liver fibrosis via TGF-b/smad signaling. (A) Rat liver samples were isolated 14 days after surgery. RNA was extracted and subjected to qRT-PCR analysis. (B) Representative images of TGFb1, TbR2, p-Smad2/3, total-Smad4, Smad7, Collagen Il and a-SMA expression detected using Western blotting. The data were quantitative of three independent experiments and are presented as mean ± S.D. (C) Representative images of TGFb1, TbR2, p-Smad2/3, total-Smad4, Smad7, Collagen I, and a-SMA expression detected using IHC. Scale bar 5 50 lm. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the model group.

Article Snippet: Next, the sections were incubated with primary rabbit antibodies against TGF-b1 (Bioswamp, dilution 1:100), TbR2 (Abcam, Cambridge, MA, USA; dilution 1:100), phosphorylated (p)-SMAD2/3 (Abcam, dilution 1:50), SMAD4 (Novus, dilution 1:100), SMAD7 (Bioswamp, dilution 1:100), collagen I and a-SMA (Abcam, dilution 1:100) overnight at 4 C. Next the sections were incubated with MaxVision horseradish peroxidase (HRP)-Polymer anti-rabbit secondary antibodies (MXB, Fujian, China) and then stained with DAB and hematoxylin following the manufacturer’s protocols.

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

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Ligustrazine Attenuates Liver Fibrosis by Targeting miR-145 Mediated Transforming Growth Factor- β /Smad Signaling in an Animal Model of Biliary Atresia.

doi: 10.1124/jpet.121.001020

Figure Lengend Snippet: Fig. 5. Ligustrazine blocked miR-145–induced TGF-b/SMAD signaling activation. (A) HSC cells were stimulated as indicated in the figure. Immu- nofluorescence was performed using a-SMA antibody (red). Nuclei were stained with 40,6-diamidino-2-phenylindole (blue). (B) The RNA levels of miR-145 and TGF-b/SMAD signaling molecules TbR2, Smad4, Smad7, and fibrosis markers Collagen I and a-SMA in each group were evaluated using qRT-PCR assay. (C) HSC cells in the 10 groups were lysed and subjected to Western blot analysis for TGF-bR2, p-Smad2/3, total Smad2/3, Smad4, Smad7, Collagen I, and a-SMA protein expression levels. The quantitative data are from three independent experiments and are pre- sented as mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the TGF-b1 treatment group; &P < 0.05, &&P < 0.01, &&&P < 0.001 compared with the TGF-b1 1 ligustrazine treatment group; $P < 0.05, $$P < 0.01, $$P < 0.001 compared with the TGF-b1 1 miR-145 knockdown treatment group.

Article Snippet: Next, the sections were incubated with primary rabbit antibodies against TGF-b1 (Bioswamp, dilution 1:100), TbR2 (Abcam, Cambridge, MA, USA; dilution 1:100), phosphorylated (p)-SMAD2/3 (Abcam, dilution 1:50), SMAD4 (Novus, dilution 1:100), SMAD7 (Bioswamp, dilution 1:100), collagen I and a-SMA (Abcam, dilution 1:100) overnight at 4 C. Next the sections were incubated with MaxVision horseradish peroxidase (HRP)-Polymer anti-rabbit secondary antibodies (MXB, Fujian, China) and then stained with DAB and hematoxylin following the manufacturer’s protocols.

Techniques: Activation Assay, Staining, Quantitative RT-PCR, Western Blot, Expressing, Knockdown