Cobalt chloride hexahydrate (CoCl2·6H2O; Sigma-Aldrich; Merck KGaA) was dissolved in deionized water to obtain a 200 mmol/l CoCl2 stock solution. The solution was further diluted with DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) to achieve the desired concentration prior to use. Rhodamine phalloidin (Invitrogen; Thermo Fisher Scientific, Inc.) was dissolved in methanol to yield a stock concentration of 200 U/ml. The solution was diluted with PBS to achieve the staining solution required for subsequent experiments. DAPI (cat. no. D1306) was purchased from Invitrogen; Thermo Fisher Scientific, Inc. Mouse anti-human fascin-1 (cat. no. TA807305), rabbit anti-human HIF-1α (cat. no. ZA0552) and mouse anti-human β-actin (cat. no. TA-09) antibodies were purchased from Origene Technologies, Inc. Mouse anti-human HIF-1α antibody (cat. no. 79233) was purchased from Cell Signaling Technology, Inc. Matrix metalloproteinase (MMP)-2 (cat. no. 10373-2-AP) and MMP-9 (cat. no. 10375-2-AP) antibodies were purchased from ProteinTech Group, Inc. E-cadherin antibody (cat. no. 610181) was purchased from BD Biosciences. Vimentin antibody (cat. no. ab92547) was purchased from Abcam.
E cadherin antibody
Manufactured by Proteintech
Sourced in China, United States
The E-cadherin antibody is a laboratory reagent used in various research applications. It is a protein that binds specifically to the E-cadherin molecule, which is involved in cell-cell adhesion. This antibody can be used to detect and study the expression and localization of E-cadherin in biological samples.
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Lab products found in correlation
Vimentin antibody, by Proteintech (5 mentions)
N cadherin antibody, by Proteintech (3 mentions)
Gapdh antibody, by Proteintech (2 mentions)
Cobalt chloride hexahydrate, by Merck Group (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Antgene (1 mentions)
Gpx4 antibody, by Proteintech (1 mentions)
Irf2 antibody, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Pv 6001, by ZSGB-BIO (1 mentions)
Anti pcna, by Abcam (1 mentions)
Protein extraction kit, by Wanlei (1 mentions)
P53 antibody, by Proteintech (1 mentions)
Ki67 antibody, by Abcam (1 mentions)
Anti caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Pcna antibody, by Proteintech (1 mentions)
13 protocols using e cadherin antibody
1
Cobalt Chloride and Cell Imaging
2
Gastric Cancer Immunostaining Quantification
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A total of 56 archived gastric cancer tissues were fixed with 4% paraformaldehyde, then paraffin-embedded, sectioned (5mm), deparaffinized, rehydrated, and antigen extracted. The tissue part was used to incubate the CAS blocking buffer and subsequently used to incubate the main antibody (E-Cadherin Antibody, Rabbit Polyclonal, PROTEINTECH, Catalog number: 20874-1-AP. Vimentin Antibody, Rabbit Polyclonal, PROTEINTECH, Catalog number: 10366-1-AP. Vinculin Antibody, Rabbit Polyclonal, Affinity Biosciences Cat# AF5122, RRID:AB_2837608). It was then overnighted at 4° C and DAKO envisio-hrp (DAKO) was used to check the reactivity afterward. By multiplying the percentage of positive cells (P) by the intensity (I), the degree of immunostaining is scored according to the following formula: H = P × I. The range of P is 0 to 4 (5% scored 0; 5-25% scored 1; 25–50% scored 2; 50–75% scored 3; 75–100% scored 4). The range of I is 0 to 3 (0, no staining; 1, weak; 2, medium; 3, strong). The staining index score was calculated by multiplying P by I, and the resulting range is 0-12. The staining index score is defined as followed: 0-3 as negative; 4-12 as positive.
3
Characterization of Protein Expression
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The treated cells were lysed on ice for 30 min, then separated by 12% SDS-PAGE and transferred to a PVDF membrane, and blocked with skim milk for 1 h. Incubate the membrane with the corresponding primary antibody overnight at 4 °C: IRF2 antibody, ACSL4 antibody, GPX4 antibody, SLC7A11 antibody, E-cadherin antibody, N-cadherin antibody, Vimentin antibody, Tubulin antibody, GAPDH antibody were purchased from Proteintech. Next, the membranes were probed with horseradish peroxidase conjugated secondary antibody (Antgene, China). Finally, immunoblots were incubated with ECL detection kit (Pierce, Rockford, IL) and visualized by imaging systems, results were quantified and analyzed by Image J.
4
Comprehensive Protein Expression Analysis
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We performed Western blotting according to the methods of a previous study. Protein lysates were prepared, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes and blotted according to standard methods using the following antibodies: RUNX1 (#4336, CST), KIT (#3074, CST), CD44 (#3570, CST), cyclin D1 (#2978, CST), c-Jun (#9165, CST), LEF1 (#2230, CST), Met (#8198, CST), c-Myc (#5605, CST), TCF1/TCF7 (#2230, CST), β-catenin (#8480, CST), E-cadherin (#3195, CST), N-cadherin (#13116, CST), Vimentin (#5741, CST), TCF8/ZEB1 (#3396, CST), AXIN1 (#2087, CST) and GAPDH (60004–1-Ig, Proteintech).
Immunohistochemistry was performed following the manufacturer’s instructions (PV-6001, ZSGB-BIO, Beijing, China) using the E-cadherin antibody (60335–1-Ig, Proteintech), N-cadherin (66219–1-Ig, Proteintech), and Vimentin (60330–1-Ig, Proteintech). One independent pathologist used software ImageJ to calculate gray values for pathological scoring.
Immunohistochemistry was performed following the manufacturer’s instructions (PV-6001, ZSGB-BIO, Beijing, China) using the E-cadherin antibody (60335–1-Ig, Proteintech), N-cadherin (66219–1-Ig, Proteintech), and Vimentin (60330–1-Ig, Proteintech). One independent pathologist used software ImageJ to calculate gray values for pathological scoring.
5
Immunohistochemical Staining Protocol for Hsp60, COX4, E-cadherin, and PCNA
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Paraffin-embedded tissues were sectioned and immersed in boiled citrate-disodium hydrogen phosphate buffer (pH=6.0) with high pressure for 5 minutes for antigen retrieval. The sections was incubated with polyclonal rabbit anti-human Hsp60 (Affinity, #AF0184,1:100), anti-COX4 (Abgent,#ALS12730,1:100), E-cadherin antibody (proteintech, # 20874-1- -AP,1:100), or anti-PCNA (Abcam, #18197, 1:200) overnight at 4°C. Sections were incubated in biotinylated secondary antibody and streptavidin peroxidase. For color development, fresh 3,3′-diaminobenzidine (DAB) solution was added to tissue slices, followed by counterstaining with hematoxylin. IHC staining was scored by two independent pathologists (Xingchun Zhou and Xiaojun Huang) who are blinded to the clinical characteristics of the patients. In brief, the percentage of positive stained cells were scored as 0 (0-9%), 1 (10%-25%), 2 (26%-50%), 3 (51%-75%) or 4 (76%-100%), and the intensity as 0 (no staining), 1 (weak staining), 2 (moderate staining) or 3 (dark staining). The total score was calculated as the product of intensity and extent, ranging from 0 to 12. A total score of < 1, ≥ 1 to < 4, ≥ 4 to <8, and ≥ 8 was defined as being negative (-), weak positive (+), moderate positive (++), and strong positive (+++), respectively.
6
Evaluating β-catenin-E-cadherin Interaction
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The Co-IP analysis was used for examination of the change in association between β-catenin and E-cadherin in the sh-CARMA3 cells. The protein was extracted from sh-CARMA3 and NC cells by Protein Extraction Kit (WLA019, Wanleibio, China), precipitated by E-cadherin antibody (20874-1-AP, Proteintech, China), and then the precipitated protein was evaluated by Western blotting using β-catenin antibody (1:2000; #2698, CST, USA).
7
Rapamycin and 5-FU Synergistic Effects on Colorectal Cancer
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Rapamycin and 5-FU were obtained from meilunbio (Dalian, China). The anti Caspase3 antibody, Cleaved Caspase3 antibody, p-AKT (ser 473) antibody, p-AKT (Thr 308) antibody, p70 S6 Kinase antibody, and p-p70S6 Kinase (Thr 389) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). The ki-67 antibody was purchased from Abcam (Cambridge, UK). The BCL-2 antibody, Bax antibody, AKT antibody, p53 antibody, E-Cadherin antibody, N-Cadherin antibody, Vimentin antibody, MMP-9 antibody and PCNA antibody were purchased from Proteintech (Wuhan, China). The GAPDH rabbit polyclonal antibody was obtained from XianZhi Biotech (Hangzhou, China). The peroxidase-conjugated secondary antibodies were obtained from CWBIO (Taizhou, China).
The human CRC cell line HCT-116 and SW-480 cells were purchased from iCell Bioscience Inc (Shanghai, China). The cells were cultured in a DMEM medium containing 10% FBS in a 37 °C incubator containing 5% CO2.
Female athymic nude mice (6–8 weeks old) were purchased from Guangdong Medical Laboratory Animal Center. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The Laboratory Animal Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University approved all experimental protocols.
The human CRC cell line HCT-116 and SW-480 cells were purchased from iCell Bioscience Inc (Shanghai, China). The cells were cultured in a DMEM medium containing 10% FBS in a 37 °C incubator containing 5% CO2.
Female athymic nude mice (6–8 weeks old) were purchased from Guangdong Medical Laboratory Animal Center. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The Laboratory Animal Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University approved all experimental protocols.
8
Isolation and Quantification of Cellular Proteins
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The total protein of cell or tissue lysate was isolated as previously described [45 (link)]. Cells were harvested in radioimmunoprecipitation (RIPA) assay buffer(Santa Cruz Biotechnology,#sc-24948). Protein content was quantified using the Bradford assay with BSA as standard (Bio-Rad), and 30μg of total protein lysate were separated on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking (5% non-fat milk in PBS, 1 h at RT), membranes were incubated at 4°C overnight with the following primary antibodies: Hsp60 antibody (Affinity, #AF0184,1:1000), β-actin monoclonal antibody (Kangchen, #KC-5A08,1:5000), AFP antibody(Abcam, #3980, 1:1000), E-cadherin antibody (proteintech,# 20874-1- -AP,1:1000), Vimentin Antibody (proteintech, #10366-1-AP,1:1000), N-Cadherin antibody (Abcam, #ab18203) and COX4 antibody (Abgent, #ALS12730,1:1000). After three washes with PBS, membranes were incubated with peroxidase-labelled secondary antibodies (Merck Millipore, 1:5000) at 37° C for 1 h. Immunoblots were developed using the enhanced chemiluminescence reagent (Pierce, #32209) .
9
Protein Expression Analysis by Western Blot
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Western blotting was performed in accordance with the standard protocols. In brief, cellular proteins were extracted using the RIPA lysis buffer, separated on SDS-PAGE gels, and transferred onto NC membranes (Millipore, Billerica, USA). After incubated in primary and second antibodies separately, proteins were detected by ECL reagent (Millipore, USA) and imaged with FluorChem Q (Protein simple, USA). The following antibodies were used for western blotting: PKM2 antibody (Proteintech, China, 1 : 1000, 15822-1-AP), vimentin antibody (Proteintech, China, 1 : 1000, 10366-1-AP), E-cadherin antibody (Proteintech, China, 1 : 1000, 20874-1-AP), N-cadherin antibody (Proteintech, China, 1 : 1000, 22018-1-AP), and GAPDH antibody (ZSGB-bio, China, 1 : 1000, TA-8).
10
Quantitative E-cadherin Immunostaining
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The paraffin-embedded tissues were collected from the Pathology Department of the Affiliated Hospital of Southwest Medical University. The tissue slides were then deparaffinized, rehydrated, and stained overnight at 4°C with a 1:500 dilution of the rabbit polyclonal E-cadherin antibody (208741AP, Proteintech). Moreover, the slides were incubated with streptavidin horseradish peroxidase (HRP) after being treated with biotinylated secondary antibody. Finally, they were stained with 3, 3’-Diaminobenzidine (DAB) and haematoxylin counterstained. Immunostaining intensity was graded as follows: 1 mild (+), 2 moderate (++), and 3 high (+++).
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