Cell lysates were separated by SDS-polyacrylamide gel (4%–10%) electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA). The membranes were then blocked with 5% skimmed milk and incubated overnight at 4 °C with the following primary detection antibodies: anti-BIN1 (14647-1-AP, Proteintech, Rosemont, USA), anti-E-cadherin (60335-1-Ig, Proteintech), anti-N-cadherin (66219-1-Ig, Proteintech), and anti-GAPDH (ab8245, Abcam). The species-matched secondary antibodies were then incubated for 2 h at room temperature and the proteins were detected using BeyoECL Plus (Beyotime, Shanghai, China). Each experiment contained triplicate wells of each sample, and all experiments were repeated at least three times.
Anti e cadherin 60335 1 ig
Manufactured by Proteintech
Sourced in United States, China, United Kingdom
Anti-E-cadherin (60335-1-Ig) is a primary antibody that binds to the E-cadherin protein. E-cadherin is a cell-cell adhesion molecule that plays a crucial role in maintaining cell-cell interactions and tissue integrity.
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Lab products found in correlation
Anti n cadherin 66219 1 ig, by Proteintech (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti vimentin 10366 1 ap, by Proteintech (2 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
Anti vimentin 60330 1 ig, by Proteintech (1 mentions)
Anti mmp2, by Proteintech (1 mentions)
Anti mmp9 10375 2 ap, by Proteintech (1 mentions)
Anti n cadherin 22018 1 ap, by Proteintech (1 mentions)
Bca protein concentration determination kit, by Beyotime (1 mentions)
Anti gapdh 60004 1 ig, by Proteintech (1 mentions)
Ab97779, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
Protease inhibitor cocktail, by GenDEPOT (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Ab184699, by Abcam (1 mentions)
6 protocols using anti e cadherin 60335 1 ig
1
Western Blot Analysis of Cell Lysates
2
Immunoprecipitation and Immunoblotting Assays
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Proteins from cells were incubated with antibody and precipitated with protein A/G-agarose beads. The immunoprecipitated proteins were subjected to SDS-PAGE. The immunoblotting assay was performed using the following antibodies: anti-N-cadherin (66219-1-Ig; Proteintech, Wuhan, China), anti-E-cadherin (60335-1-Ig; Proteintech), anti-MMP9 (10375-2-AP; Proteintech), anti-MMP2 (66366-1-Ig; Proteintech), anti-vimentin (60330-1-Ig; Protein tech), anti-PKM2 (A0540; ABclonal, Wuhan, China), anti-COX-2 (12282; CST, MA, United States), anti-ERK1/2 (sc-292838; Santa Cruz, CA, United States), anti-p-ERK1/2 (AP0472; ABclonal), anti-β-actin (RM2001; Ray, Beijing, China), and anti-α-tubulin (RM2007; Ray).
3
Protein Expression Analysis in Cell Lines
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Total protein extraction of cell lines was conducted using radio immunoprecipitation assay (RIPA) buffer and phosphatase inhibitors (Roche, Sigma, St. Louis MO, USA). Subsequently, we used the BCA protein concentration determination kit (Beyotime Institute of Biotechnology) to quantitate the protein concentration. Extracted proteins were resolved by SDS-PAGE and transferred to 0.22 μm PVDF membranes. The membranes were incubated with the corresponding primary antibody at 4 °C for 16 h, followed by secondary antibody with 1 h at room temperature. Finally, the protein bands were presented using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The relevant antibodies used for this study were antiNEK2 (sc55601, Santa Cruz Biotechnology), anti-E-cadherin (60335-1-Ig, Proteintech), anti-Vimentin (10366-1-AP, Proteintech), anti-N-cadherin (22018-1-AP, Proteintech), anti-MMP2 (ab97779, Abcam), anti-MMP9 (ab76003, Abcam), β-catenin (ab32572, Abcam), anti-cyclin D1 (1:200 dilution, ab16663, Abcam), anti- c-Myc (ab32072, Abcam), anti-GAPDH (60004-1-Ig, Proteintech).
4
Western Blot Analysis of Cell Signaling
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Cells were lysed with RIPA lysis buffer containing protease inhibitor cocktail (GenDEPOT, Harris County, TX, USA) for 20 min on ice. The lysate was centrifuged at 12,000 rpm for 30 min at 4°C. The protein concentrations were measured using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skim milk at room temperature for 1 h, then incubated with primary antibodies overnight at 4°C. After washing extensively, the membrane was incubated with HRP-conjugated secondary antibody for 2 h. The signal was detected using enhanced chemiluminescence system.
The primary antibody information used in this study is as follows: anti-KIF2A (ab197988), anti-GAPDH (ab8245), anti-N-cadherin (ab18203), anti-p-Akt (ab38449), anti-Akt (ab8805), anti-p-ERK (ab79483), anti-ERK (ab184699), anti-p-JNK (ab4821), anti-JNK (ab112501), anti-p-c-Jun (ab32385), and anti-c-Jun (ab40766) were purchased from Abcam; anti-E-cadherin (60335-1-Ig) and anti-Vimentin (10366-1-AP) were purchased from Proteintech.
The primary antibody information used in this study is as follows: anti-KIF2A (ab197988), anti-GAPDH (ab8245), anti-N-cadherin (ab18203), anti-p-Akt (ab38449), anti-Akt (ab8805), anti-p-ERK (ab79483), anti-ERK (ab184699), anti-p-JNK (ab4821), anti-JNK (ab112501), anti-p-c-Jun (ab32385), and anti-c-Jun (ab40766) were purchased from Abcam; anti-E-cadherin (60335-1-Ig) and anti-Vimentin (10366-1-AP) were purchased from Proteintech.
5
Western Blot Analysis of Cellular Signaling
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After culture and treatments, cells were harvested and lysed in protein extraction reagent (78501, ThermoFisher) and mixed with 1% protease inhibitor mixture at 4 °C for 30 min. Protein samples (40 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Next, the membranes were blocked with 10% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies: anti-IDH3α (15909-1-AP, Proteintech, Chicago, IL, USA), anti-HIF-1α (ab51608, Abcam, Cambridge, UK), anti-E-cadherin (60335-1-Ig, Proteintech), anti-N-cadherin (66219-1-Ig, Proteintech), anti-Vimentin (ab8978, Abcam), anti-slug (12129-1-AP, Proteintech), anti-cGAS (15102, CST, Danvers, MA, USA), anti-sting (13647, CST), anti-phospho-sting (Ser366) (19781,CST), anti-TBK1 (38066, CST), anti-phospho-TBK1 (S172) (ab109272, Abcam), anti-IRF3(ab76409, Abcam), anti-phospho-IRF3(S386)(ab76493, Abcam), anti-p65 (ab32536, Abcam), anti-phospho-p65 (S536) (ab76302, Abcam), anti-PD-L1 (ab213524, Abcam), and β-tubulin (sc-8432, Proteintech). Finally, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Western blotting analysis was performed using a Bio-Rad chemiluminescence imager. Original blots can be found at Supplementary Figure S5 .
6
Profiling of Hepatocellular Carcinoma Cells
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Human HCC cells (HepG2 and Huh7) were purchased from American Type Culture Collection (Manassas, VA). Human HCC cells (Hep3B, SMMC-7721, and HCC-LM3) and immortalized liver cell line (HL-7702) were kindly provided by the Department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. Thirty-two HCC samples and matched non-tumor tissues were consecutively collected from patients undergoing curative resection between September 2005 and May 2019 at the Department of General Surgery, The First College of Clinical Medical Science, China Three Gorges University, Yichang Central People’s Hospital (Table S1 ). miR-30c-5p inhibitor, its control (referred to as anti-miR Ctrl), miR-30c-5p mimic, and corresponding negative control were purchased from RiboBio (Guangzhou, China). The primary antibodies used in this work were as follows: anti-E-cadherin (60335-1-Ig) was obtained from Proteintech Group (Chicago, IL); anti-RAB32 (ab251764) and anti-Ki67 (ab15580) were purchased from Abcam; and anti-β-actin (3700S), anti-N-cadherin (13116S), anti-Snail (3879P), anti-Vimentin (5741P), and anti-β-catenin (8480P) were obtained from Cell Signaling Technology (Beverly, MA).
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