anti tgf β2 polyclonal goat igg antibody  (R&D Systems)

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: GO and KEGG pathway enrichment analyses of DEGs in MCF-7 cells after GGCT knockdown. (A) GO enrichment analysis for biological processes (up-regulated and down-regulated genes), and KEGG pathway analysis of DEGs, using samples collected 3 days after knockdown. The top 10 biological process terms ranked according to p-Values are shown. (B) Hypothetical schema of cell cycle regulation following GGCT knockdown in MCF-7 cells, constructed based on DEGs enriched in the KEGG pathway ‘hsa04110: Cell cycle’ (http://www.kegg.jp). (C) Relative mRNA expression patterns of GGCT, TGF-β2, CDKN1A (p21 Cip1 ), and CDKN2B (p15 INK4b ) were measured with qRT-PCR. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using two-tailed Student’s t-test for pairwise comparisons. CDKN1A, Cyclin dependent kinase inhibitor 1A; CDKN2B, cyclin dependent kinase inhibitor 2B; DEGs, differentially expressed genes; GGCT, γ-glutamylcyclotransferase; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; TGF-β2, transforming growth factor-β2.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Knockdown, Construct, Expressing, Quantitative RT-PCR, Two Tailed Test, Reverse Transcription, Polymerase Chain Reaction

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: TGF-β2/SMAD signaling axis regulates p15 INK4b and p21 Cip1 expression in GGCT-depleted MCF-7 cells. (A) mRNA expression levels of GGCT and TGF-β2 were analyzed by qRT-PCR 3 days after transfection with GGCT- and/or TGF-β2-siRNA. (B) Western blot analysis of GGCT and TGF-β2 expression 4 days after transfection with GGCT- and/or TGF-β2-siRNA. (C) Western blotting analysis of p15 INK4b , p21 Cip1 , phospho-SMAD2 (pSMAD2), SMAD2, phospho-SMAD3 (pSMAD3), SMAD3, GGCT, and α-tubulin in MCF-7 at 4 days after transfection with GGCT-siRNA and/or TGF-β2-siRNA, or non-target control siRNA. (D) Western blotting analysis of p15 INK4b , p21 Cip1 , pSMAD3, SMAD3, GGCT, and α-tubulin in MCF-7 cells at 4 days after transfection with GGCT-siRNA and/ or SMAD3-siRNA, or non-target control siRNA. (E) The number of viable cells in the trypan blue dye exclusion test and (F) representative images of MCF- 7 cells at 4 days after transfection with the indicated siRNAs. Scale bar: 200 μm; n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 vs. control, †p<0.05; ††p<0.01, and †††p<0.001 vs. GGCT, one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; pSMAD, phosphorylated SMAD; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: p15 INK4b , p21 Cip1 , and their upstream regulator TGF-β2 are involved in the induction of cellular senescence following GGCT-KD in MCF-7 cells. (A) Representative images of SA-β-Gal staining 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT, p15, and p21. Scale bar: 50 μm. (B) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. (C) Representative images of SA-β-Gal staining at 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT and TGF-β2. Scale bar: 50 μm. (D) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. n=3 per group; *p<0.05, **p<0.01, ***p<0.001, using one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; KD, knockdown; SA-β-Gal, senescence-associated β-galactosidase; siRNA, smallinterfering RNA; TGF-β2, transforming growth factor-β2.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Staining, Transfection, Knockdown