All antibodies used for western blot analysis were 1:1000 dilution except for CK2α (1:500) and GAP (1:2000) and are as follows: From Cell Signaling Technology (CST); BRD4 (13,440), AXL (8661S), E-cadherin (3195), N-cadherin (13,116), β-catenin (8480), Vimentin (5741), EGFR (2232S), Ras (8955S), EGFR (L858R mutant specific) (3197), MYC (5605), and FOSL1 (5281). From abcam; CK2α (ab137788), and from Santa Cruz; GAP (sc-47724). Lastly the pBRD4 antibody was a gift from C. M. Chiang with specificities as previously described44 (link) and used to detect pBRD4 as described in19 (link). (+)-JQ1 (active enantiomer) (cat. # 4499) was purchased from TOCRIS. I-BET762 (cat. # 10676) was purchased from Caymen Chemical. Crizotinib (cat. # S1068) and CX-4945 (also known as Silmitasertib) (cat# S2248) were purchased from Selleck Chemical, and BEZ235 (cat. #CT-BEZ) was purchased from ChemieTEK. All drugs were suspended in DMSO. Recombinant human TGF-beta 1 (TGFβ1) protein was purchased from R & D Systems (cat. # 240-B-002) and resuspended in 4 mM HCl containing 1 mg/mL BSA to a final concentration of 10 μg/mL. This was then added to regular cultured media at 1:1000 to give a final concentration of 10 ng/mL.
E cadherin 3195
Manufactured by Cell Signaling Technology
Sourced in United States
E-cadherin #3195 is a primary antibody product manufactured by Cell Signaling Technology. It is designed for the detection of E-cadherin protein expression in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin 5741, by Cell Signaling Technology (6 mentions)
N cadherin 13 116, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Snail, by Cell Signaling Technology (3 mentions)
β catenin, by Cell Signaling Technology (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Molecular weight standards, by Thermo Fisher Scientific (2 mentions)
Api a3145, by Merck Group (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
S2367, by Agilent Technologies (2 mentions)
K3468, by Agilent Technologies (2 mentions)
Pk6106, by Vector Laboratories (2 mentions)
Inhibin βa hpa020031, by Merck Group (2 mentions)
Dp72 8 bit rgb camera, by Olympus (2 mentions)
X0909, by Agilent Technologies (2 mentions)
Zeb1 3396, by Cell Signaling Technology (2 mentions)
Gapdh fl 335, by Santa Cruz Biotechnology (2 mentions)
Bip 3183, by Cell Signaling Technology (2 mentions)
Eif2α 9722, by Cell Signaling Technology (2 mentions)
27 protocols using e cadherin 3195
1
Western Blot Antibody Dilutions and Reagents
2
Investigating Signaling Pathways in Cancer
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Antibodies specific to EphA2 (ab118882), YES1 (ab109744), TAZ (ab119253), and GAPDH (ab37168) and horseradish peroxidase‐couple secondary antibodies were purchased from Abcam. Antibodies targeting TEAD2 (8870), E‐cadherin (3195), N‐cadherin (13116), and vimentin (5741) were from Cell Signaling Technology. miR‐125a‐5p mimics, inhibitors (antagomir), negative controls, 3′‐untranslated region (UTR) reporter plasmids, small hairpin RNA (shRNA) specifically targeting TAZ, and control shRNA were synthesized by Ribobio Co. The sequences are shown in Table S1 .
3
Immunohistochemical Analysis of PDAC Biomarkers
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Immunohistochemistry was performed as described earlier [28 (link),29 (link)]. In brief, a human PDAC tissue microarray (PA484, US Biomax, Rockville, MD, USA) and HPAC cell line xenograft tissues were used to assess the expression of SOD2 (13141), pAkt (4060), BAX (2772), Snail (3879), cleaved caspase 9 (9509), pmTOR (5536), and E-cadherin (3195) proteins (Cell signaling Technology Inc., Danvers, MA, USA). The experimental conditions and detailed protocols are described in the Supplementary Materials .
4
Colorectal Cancer Cell Culture and Protein Expression
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The SW480 and HT-29 cells were purchased from the cell bank of Shanghai Institutes for Biological Sciences (Shanghai, China). SW480 were cultured in RPMI 1640 (Hyclone, Logan, TX, USA) and HT-29 were cultured in DMEM (Gibco, Brookyln, NY, USA) with 5% CO2 in 37 °C. All media were supplemented with 10% Fetal Bovine Serum (16000-044, Gibco, Brookyln, NY, USA) and 1% penicillin/streptomycin. The main antibodies including purchased from PHF19 (77271), Cyclin D1 (55506), E-cadherin (3195), N-cadherin (13116), β-catenin (8480), and Twist (46702) were purchased from Cell Signaling Technology (Beverly, MA, USA); H3K27me3 (ab6002) was purchased from Abcam (Cambridge, UK); CDK4 (11026-1-AP), CDK6 (14052-1-AP), Tubulin (11224-1-AP), and GADPH (10494-1-AP) were purchased from Proteintech (Wuhan, Hubei, China).
5
Comprehensive Materials and Reagents for Cell Assays
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API (A3145) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS), antibiotics, molecular weight standards, trypsin-EDTA, and all medium additives were obtained from Life Technologies (Gaithersburg, MD). The CellTiter 96 AQueous One Solution Proliferation Assay System was purchased from Promega (Madison, WI). An enhanced chemiluminescence (ECL) kit was purchased from Amersham (Arlington Heights, IL). Antibodies specific for fibronectin (ab2413) and SPOCK1 (ab229935) were obtained from Abcam (Cambridge, MA). Antibodies specific for cleaved-PARP (#9541), phospho-Ak (ser473, #9271), Akt (#4691), Snail (#3895), Slug (#9585), Twist (#46702), and E-cadherin (#3195) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies specific for vimentin (550513) and N-cadherin (610920) were purchased from BD Biosciences (San Jose, CA). Antibodies specific for cyclin D1 (sc-8396), cyclin E (sc-377,100), β-actin (sc-47,778), and goat anti-rabbit (sc-2004) and anti-mouse (sc-2005) immunoglobulin G (IgG) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene fluoride (PVDF) membranes for Western blotting were purchased from Bio-Rad (Hercules, CA). Unless otherwise specified, other chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO).
6
Protein Extraction and Western Blot
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Proteins were extracted in RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail and a phosphatase inhibitor (Bimake, USA) and determined by the BCA assay kit (Thermo, USA). After polyacrylamide gel electrophoresis, proteins were transferred to a PVDF membrane and incubated overnight at 4°C with specific primary antibodies. Antibodies against PARK2 (ab15954), CDK2 (ab32147), mTOR (ab2732), p-mTOR (ab109268), EGFR (ab52894), p-EGFR (ab40815), and AKT (ab8805) were purchased from Abcam (Cambridge, MA, USA). E-cadherin (3195), N-cadherin (13116), vimentin (5741), slug (9585) claudin-1 (13255), cyclin D1 (2978), CDK4 (12790), cyclin D3 (2936), P21 (2947), P18 (2896), cleaved caspase-3 (9664), cleaved caspase-8 (8592), cleaved caspase-9 (20750), cleaved PARP (5625), Cytochrome c (11940), p-AKT (4060), and GAPDH (2118) were obtained from Cell Signaling Technology (Boston, USA). The goat anti-rabbit IgG–HRP (HA1001-100) and goat anti-mouse IgG–HRP (HA1006) were obtained from HuaAn Biotechnology (Huabio, China). Then the PVDF membrane was incubated with secondary antibody at room temperature for 1 h. The membranes were observed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo, USA). Band intensity was analyzed with Quantity One.
7
Mucosal Protein Profiling via Western Blot
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Western blot analysis was carried out with standard methods on the mucosal protein lysates obtained from each mouse. Specific antibodies against p-Stat3 (#9138), Stat3 (#9132), p-Src (#6943), Src (#2123), N-Cadherin (#4061), E-Cadherin (#3195), arginase-1 (#9819), and iNOS (#2982) were purchased from Cell Signaling Technology (Beverly, MA). Anti-FOXP3 antibody (ab54501) was purchased from Abcam (Cambridge, UK) and anti-α-tubulin (sc-20357) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
8
Immunohistochemical Analysis of Breast Tissue Microarrays
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Assembly and IHC staining of human breast tissue microarrays (TMAs) were performed by the Biorepository and Tissue Analysis core at MUSC. TMAs consisted of matched normal and tumor tissue as well as normal and metastatic lymph node biopsies from patients for which survival data was available (Supplementary Table S2 ). For IHC staining of formalin-fixed, paraffin-embedded TMAs and mouse lung tissue, sections were deparaffinized in xylene, rehydrated in alcohol, and processed as follows: The sections were incubated with target retrieval solution (S2367; Dako, Carpinteria, CA, USA) in a steamer for 45 min followed by 3% hydrogen peroxide solution for 10 min and protein block (X0909; Dako) for 20 min at room temperature. Sections were incubated overnight in a humid chamber at 4°C with antibodies against Inhibin βA [HPA020031] (1:3200; Sigma), E-cadherin [#3195], Vimentin [#5741] or Ki-67 [#12202] (1:400; Cell Signaling) followed by biotinylated secondary antibody (PK6106; Vector laboratories, Burlingame, CA, USA) for 30 min and ABC reagent for 30 min. Immunocomplexes of horseradish peroxidase were visualized by DAB reaction (K3468; Dako), and sections were counterstained with hematoxylin before mounting. Micrographs of stained sections were taken using an Olympus DP72 8-bit RGB camera with Cellsens acquisition software.
9
Epithelial-Mesenchymal Transition Markers
10
Comprehensive Immunoblotting Approach for EMT
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Rabbit monoclonal anti-ZEB1 (3396, s; clone D80D3), cleaved NOTCH1 (4147, s; clone D3B8; specific for detecting NICD1), RBPJ (5313, s; clone D10A4), E-cadherin (3195, s; clone 24E10), Vimentin (5741, s; clone D21H3), Tubulin (2125, s; clone 11H10), pERBB3 (2842, s; clone D1B5) and ERBB3 (12708, s; clone D22C5) were purchased from Cell Signaling and used at a 1:3,000 dilution for western blotting. Goat polyclonal anti-Actin (sc-1616) was purchased from Santa Cruz and used at a 1:1,000 dilution for western blotting. Rabbit polyclonal anti-NOTCH1 (ab27526) was purchased from Abcam and used at a 1:3,000 dilution for western blotting. Mouse monoclonal anti-FLAG (F1804; monoclonal; clone M2) was purchased from Sigma and used at a 1:5,000 dilution for western blotting. siRNAs were purchased from Santa Cruz (pooled siRNAs) or Origene (individual siRNAs for ZEB1 and RBPJ). All short hairpin RNAs and chemicals were purchased from Sigma, except for BMS-708163 and gefitinib, which were purchased from Selleckchem. Control GFP (LPP-EGFP-LV105-025) and ZEB1 lentiviral particles (LPP-F0876-Lv105-200-S) were purchased from Genecopoiea.
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