Smad4 b 8

Manufactured by Santa Cruz Biotechnology

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Smad4 (B-8) is a primary antibody that detects the Smad4 protein. Smad4 is a key mediator of transforming growth factor-beta (TGF-β) signaling pathways. It functions as a co-Smad, forming complexes with receptor-activated Smads to regulate the transcription of target genes.

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SMAD4 (54 citations) Anti-Smad4 (B-8, (4 citations) Anti-SMAD4 mouse monoclonal antibody (clone B-8; (3 citations) Smad4 monoclonal antibody (B-8) (2 citations)

7 protocols using smad4 b 8

Antibody Panel for Signaling Pathway Analysis

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The following primary antibodies were from Cell Signaling Technology (Boston, MA): phospho-SMAD2^Ser465/467 (138D4), SMAD2/3 (D7G7), phospho-STAT3^Tyr705 (D3A7), STAT3 (79D7), phospho-AKT^Ser473 (D9E), AKT(C67E7), Phospho-EGF Receptor Tyr1045, Tyr1068 (D7A5), and Tyr1173 (53A5), EGF Receptor (D38B1), Phospho-p44/42 MAPK^{Thr202/Tyr204}, p44/p42 MAPK (137F5), GAPDH (14C10), Phospho-JNK^{Thr183/Tyr185} (81E11), JNK (56G8), BCL-2 (50E3), BCL-XL (54H6), BIM (C34C5), beta-Actin (13E5). SMAD4 (B-8) was from Santa Cruz Biotechnology (Dallas, TX).

Ozawa H., Ranaweera R., Izumchenko E., Makarev E., Zhavoronkov A., Fertig E.J., Howard J.D., Markovic A., Bedi A., Ravi R., Perez J., Le Q.T., Kong C.S., Jordan R.C., Wang H., Kang H., Quon H., Sidransky D, & Chung C.H. (2017). SMAD4 loss is associated with cetuximab resistance and induction of MAPK/JNK activation in head and neck cancer cells. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(17), 5162-5175.

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Western Blot Analysis of Protein Markers

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Cells were lysed in RIPA buffer containing a protease inhibitor. Equal amounts of protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Millipore). The membranes were incubated overnight with a primary antibody against USP3 (1:200 dilution, ab101473, Abcam, USA), SMAD4 (1:200 dilution, Smad4(B-8), Santa Cruz, USA) or β-actin (1:3000 dilution, AC-15, Sigma, USA). Afterwards, goat anti-mouse IgG or goat anti-rabbit IgG (ZSGB-BIO) was used as a secondary antibody. An ECL detection kit (Amersham, Little Chalfont, UK) was used to detect the signal. The ImageJ software (NIH, USA) was used for quantification.

Wang Z., Yang J., Di J., Cui M., Xing J., Wu F., Wu W., Yang H., Zhang C., Yao Z., Zhang N., Jiang B, & Su X. (2017). Downregulated USP3 mRNA functions as a competitive endogenous RNA of SMAD4 by sponging miR-224 and promotes metastasis in colorectal cancer. Scientific Reports, 7, 4281.

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Western Blot Analysis of Liver Cancer Cells

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RIPA lysis buffer was used to lyse liver cancer cells, and 30 μg of protein was run on SDS/PAGE gel. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked for 1 h, rinsed with TBST three times, and then incubated in the corresponding primary antibody in a cold room at 4°C overnight. The membranes were rinsed with TBST three times again and incubated with secondary antibodies. Finally, images of the membrane were taken by the ECL system (Thermo Fisher Scientific). Information about the antibodies we used in the study is listed below: MMP-9 (E-11, Santa Cruz Biotechnology), E2F3 (Thermo Fisher Scientific), CD44 (EPR18668, Abcam), SOX9 (E-9, Santa Cruz Biotechnology), GSN (H-5, Santa Cruz Biotechnology), FOXC2 (G-7, Santa Cruz Biotechnology), SMAD4 (B-8, Santa Cruz Biotechnology), α-tubulin (TU-02, Santa Cruz Biotechnology).

Liu G., Ouyang X., Gong L., Yao L., Liu S., Li J., Zhang Q, & Xiao Y. (2021). E2F3 promotes liver cancer progression under the regulation of circ-PRKAR1B. Molecular Therapy. Nucleic Acids, 26, 104-113.

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Detailed Protocol for FOXP3 and TGF-β Signaling

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The primers used in this study were listed in Supplementary Table S6. The pcDNA3.1-FOXP3, pcDNA3.1, Flag-pcDNA3.1-FOXP3, Flag-pcDNA3.1 were produced in our Lab. CS2-flag-Smad2 (Addgen plasmid #14042) and CS2-flag-Smad3 (Addgen plasmid #14052) was a gift from Joan Massague, pcDNA3-Flag-Smad4 (Addgen plasmid #80888) was a gift from Aristidis Moustakas, Pbv-Luc (Addgen plasmid #16539) and c-Myc promoter del-1 (Addgen plasmid #16601) was a gift from Bert Vogelstein. SiRNA-FOXP3 (sc-43569) and siRNA-Control (sc-43569) were from Santa Cruz Biotechnology. The primary antibodies for western blot were as follows: FOXP3 (150D/E4, Invitrogen, 1:1000), FOXP3Δ3 (16J4G6, Novus,1:1000), PCNA (PC10, Santa Cruz, 1:2000), Bax (B-9, Santa Cruz, 1:1000), Bcl2 (C-2, Santa Cruz, 1:1000), p53 (DO-1, Santa Cruz, 1:1000), Smad2 (A-11, Santa Cruz, 1:1000), Smad3 (38-Q, Santa Cruz, 1:1000), Smad2/3 (A-3, Santa Cruz, 1:1000), p-Smad2(s467, Abcam, 1:1000), p-Smad3(1D9, Santa Cruz, 1:1000), Smad4 (B-8, Santa Cruz, 1:1000), c-Myc (9E10, Santa Cruz, 1:1000), and GAPDH (6C5, Santa Cruz, 1:2000). The primary antibodies for immunohistochemistry were listed below: FOXP3 (150D/E4, Invitrogen, 1:100), FOXP3Δ3 (16J4G6, Novus,1:100), Ki-67 (Ki67, Santa Cruz, 1:50), c-Myc (9E10, Santa Cruz, 1:100), Smad2/3 (A-3, Santa Cruz, 1:100), and p-Smad3 (Santa Cruz, 1:100).

Gong Z., Jia H., Yu J., Liu Y., Ren J., Yang S., Hu B., Liu L., Lai P.B, & Chen G.G. (2020). Nuclear FOXP3 inhibits tumor growth and induced apoptosis in hepatocellular carcinoma by targeting c-Myc. Oncogenesis, 9(10), 97.

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TGF-β Signaling Pathway Analysis

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We used antibodies against total SMAD2 (D43B4, #5339) and pSMAD2 (Ser465/467, 138D4, #3108) from Cell Signaling, SMAD4 (B‐8, #sc‐7966) and TGFβRII (E‐6, #sc‐17792) from Santa Cruz, and GAPDH (#G9545) from Sigma‐Aldrich. Recombinant human TGFβ 1 was obtained from R&D Systems (#240‐B‐002) and stored at −80°C in 4 mM HCl, 1 mg/ml bovine serum albumin at 390 nM. DRB (5,6‐dichlorobenzimidazole 1‐β‐D‐ribofuranoside) was purchased from Cayman (used at 100 μM), TGFβRI kinase inhibitor VI SB431542 from Calbiochem (used at 10 μM) and CDK1 inhibitor RO 3306 (used at 3 μM) from Axon.

Strasen J., Sarma U., Jentsch M., Bohn S., Sheng C., Horbelt D., Knaus P., Legewie S, & Loewer A. (2018). Cell‐specific responses to the cytokine TGFβ are determined by variability in protein levels. Molecular Systems Biology, 14(1), e7733.

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Immunohistochemical Profiling of Tissue Samples

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Hematoxylin (Sigma MHS16) and eosin (Sigma HT110216) staining and immunohistochemistry was performed on tissues fixed in 10% neutral buffered formalin (NBF) for 24–48 hours, embedded in paraffin, and sectioned at 5 μm thickness. Immunohistochemical staining was optimized using the Discovery XT System (Ventana, Tuscon, AZ). The following antibodies were used for immunohistochemistry: β-catenin (polyclonal, Cell Signaling Technology, Danvers, MA), Ki67 (SP6, Spring Biosciences, Pleasanton, CA), cyclin D1 (SP4, Thermo Fisher Scientific, Halethorpe, MD), AR (EPR1535(2), Abcam, Cambridge, MA), cytokeratin 14 (polyclonal, BioLegend, San Diego, CA), cytokeratin 8 (polyclonal, Spring Biosciences, Pleasanton, CA), p63 (D2K8X, Cell Signaling Technology, Danvers, MA), and Smad4 (B-8, Santa Cruz Biotechnology, Dallas, TX).

Valkenburg K.C., De Marzo A.M, & Williams B.O. (2017). Deletion of tumor suppressors adenomatous polyposis coli and Smad4 in murine luminal epithelial cells causes invasive prostate cancer and loss of androgen receptor expression. Oncotarget, 8(46), 80265-80277.

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Molecular Pathways in Transcriptional Regulation

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The primers used in this study were listed in Supplementary Table S1. The pcDNA3.1-FOXP3, pcDNA3.1, Flag-pcDNA3.1-FOXP3, Flag-pcDNA3.1 were produced in our Lab. CS2-ag-Smad2 (Addgen plasmid #14042) and CS2-ag-Smad3 (Addgen plasmid #14052) was a gift from Joan Massague, pcDNA3-Flag-Smad4 (Addgen plasmid #80888) was a gift from Aristidis Moustakas, Pbv-Luc (Addgen plasmid #16539) and c-Myc promoter del-1 (Addgen plasmid #16601) was a gift from Bert Vogelstein. SiRNA-FOXP3 (sc-43569) and siRNA-Control (sc-43569) were from Santa Cruz Biotechnology. The primary antibodies for western blot were as follows: FOXP3 (150D/E4, Invitrogen, 1:1000), FOXP3Δ3 (16J4G6, Novus,1:1000), PCNA (PC10, Santa Cruz, 1:2000), Bax (B-9, Santa Cruz, 1:1000), Bcl2 (C-2, Santa Cruz, 1:1000), Smad2 (A-11, Santa Cruz, 1:1000), Smad3 (38-Q, Santa Cruz, 1:1000), Smad2/3 (A-3, Santa Cruz, 1:1000), p-Smad2(s467, Abcam, 1:1000), p-Smad3(1D9, Santa Cruz, 1:1000), Smad4 (B-8, Santa Cruz, 1:1000), c-Myc (9E10, Santa Cruz, 1:1000), and GAPDH (6C5, Santa Cruz, 1:2000). The primary antibodies for immunohistochemistry were listed below: FOXP3 (150D/E4, Invitrogen, 1:100), FOXP3Δ3 (16J4G6, Novus,1:100), Ki-67 (Ki67, Santa Cruz, 1:50), c-Myc (9E10, Santa Cruz, 1:100), Smad2/3 (A-3, Santa Cruz, 1:100), and p-Smad3 (Santa Cruz, 1:100).

Gong Z., Hao J., Yu J., Liu Y., Ren J., Yang S., Hu B., Liu L., Lai P.B., & Chen G.G. (2020). FOXP3 inhibits tumor growth and induced apoptosis in hepatocellular carcinoma by targeting c-Myc.

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