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3 protocols using smad9

1

Antibody-Mediated Protein Interactome Analysis

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Antibodies used include: GSDMD Rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), GSDMD Rabbit monoclonal Ab (Abcam, Ab209845), FLAG® M2 monoclonal Ab (Sigma, F3165), CARD8 C terminus Rabbit polyclonal Ab (Abcam, Ab24186), CARD8 N-terminal Rabbit polyclonal Ab (Abcam, Ab194585), GAPDH Rabbit monoclonal Ab (Cell Signaling Tech, 14C10), MTMR1 (Abcam, Ab 240569), D2HGDH (Abcam, Ab233516), ATF-3 (Cell Signaling Tech, D2Y5W), IGBP1 (Cell Signaling Tech, 5F6), SMAD9 (Abcam, Ab124094), ARF1 (Abcam, Ab58578), ACTR5 (Proteintech, 21505), AKAP8 (Abcam, Ab72196), GSTP1 (Cell Signaling Tech, 3F2), SET (Abcam, Ab1183), NLRP1B (Vance laboratory, 2A12), IRDye 800CW anti-rabbit (LICOR, 925-32211), IRDye 800CW anti-mouse (LI-COR, 925-32210), IRDye 680CW anti-rabbit (LI-COR, 925-68073), IRDye 680CW anti-mouse (LI-COR, 925-68072). Other reagents used include: Val-boroPro (VbP) (Okondo et al., 2017 (link)), Bortezomib (Bort; MilliporeSigma, 504314), MLN4924 (Cayman, 15217), bestatin methyl ester (Me-Bs; Sigma, 200485), sequencing grade modified trypsin (Promega, V5113), proteinase K (PROK; Invitrogen, 25530049), FuGENE HD (Promega, E2311), TMTsixplex Isobaric Label Reagents (ThermoFisher Scientific).
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2

Antibody-Mediated Protein Interactome Analysis

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Antibodies used include: GSDMD Rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), GSDMD Rabbit monoclonal Ab (Abcam, Ab209845), FLAG® M2 monoclonal Ab (Sigma, F3165), CARD8 C terminus Rabbit polyclonal Ab (Abcam, Ab24186), CARD8 N-terminal Rabbit polyclonal Ab (Abcam, Ab194585), GAPDH Rabbit monoclonal Ab (Cell Signaling Tech, 14C10), MTMR1 (Abcam, Ab 240569), D2HGDH (Abcam, Ab233516), ATF-3 (Cell Signaling Tech, D2Y5W), IGBP1 (Cell Signaling Tech, 5F6), SMAD9 (Abcam, Ab124094), ARF1 (Abcam, Ab58578), ACTR5 (Proteintech, 21505), AKAP8 (Abcam, Ab72196), GSTP1 (Cell Signaling Tech, 3F2), SET (Abcam, Ab1183), NLRP1B (Vance laboratory, 2A12), IRDye 800CW anti-rabbit (LICOR, 925-32211), IRDye 800CW anti-mouse (LI-COR, 925-32210), IRDye 680CW anti-rabbit (LI-COR, 925-68073), IRDye 680CW anti-mouse (LI-COR, 925-68072). Other reagents used include: Val-boroPro (VbP) (Okondo et al., 2017 (link)), Bortezomib (Bort; MilliporeSigma, 504314), MLN4924 (Cayman, 15217), bestatin methyl ester (Me-Bs; Sigma, 200485), sequencing grade modified trypsin (Promega, V5113), proteinase K (PROK; Invitrogen, 25530049), FuGENE HD (Promega, E2311), TMTsixplex Isobaric Label Reagents (ThermoFisher Scientific).
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3

Analyzing Tumor Stem Cell Markers

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The anti-human B7-H3, MMP2 and MMP9 antibodies used for immunohistochemistry were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States). The anti-human B7-H3, E-Cadherin, N-Cadherin, Vimentin, β-catenin, CD44, Oct4, Akt, phosphorylated Akt, Smad1 and Smad9 antibodies were from Abcam (Cambridge, MA, United States). The anti-human phosphorylated Smad1/5 and Smad1/5/9 antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, United States). The anti-human CD133 antibody for flow cytometry was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The horseradish peroxidase-conjugated secondary anti-mouse and anti-rabbit antibodies and the GAPDH antibody were from Beyotime (Nantong, China). The fluorescent dye DiI that was used to label the cell membrane was from Keygentec (Nanjing, China). The Jak2-specific inhibitor AG490 was purchased from Sigma-Aldrich (St. Louis, MO, United States). The PI3K-specific inhibitor LY294002 was from Selleckchem (Houston, TX, United States). The MatrigelTM matrix used in the invasion assay was from BD (San Jose, CA, United States).
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