Protein samples were separated by electrophoresis on 12% SDS-PAGE gels and transferred to PVDF membranes (GE Healthcare). The western blot procedure was performed as described previously (Shanmugasundaram et al., 2017 (link)) with minor modifications. Briefly, membranes were blocked for 1 h in 5% non-fat milk, 1× TBS, 0.1% Tween-20 and probed with the corresponding primary and secondary antibodies. For detection of PRDX6 protein the membrane was incubated in 5% non-fat milk, 1× TBS, 0.1% Tween-20 with 1:1,000 polyclonal rabbit anti-PRDX6 antibody (ab59543, Abcam) 1.5 h at room temperature followed by incubation for 1 h at room temperature with 1:15,000 hP-conjugated anti-rabbit secondary antibody (ab6721, Abcam). Beta-actin as a loading control was detected by incubation in 5% non-fat dry milk, 1× TBS, 0.1% Tween-20 with 1:10,000 anti-β-Actin antibody (4967S, Cell Signalling) 1.5 h at room temperature followed by incubation for 1 h at room temperature in 1:20,000 anti-rabbit polyclonal antibody (ab6721, Abcam). The signals were quantified using ImageJ 1.52a (NIH) software based on the calculation of peak area and the intensity values for PRDX6 bands were normalized across the samples by the actin control. Statistical significance of differences between the group means was evaluated by ANOVA with Tukey correction for multiple comparisons in Prism 6 (GraphPad Software).
Ab6721
Manufactured by Abcam
Sourced in United States, United Kingdom, China, France, Japan, Hong Kong, Germany
Ab6721 is an anti-rabbit IgG antibody conjugated to Alexa Fluor 488. It is designed for use in immunofluorescence applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab9485, by Abcam (138 mentions)
Pvdf membrane, by Merck Group (137 mentions)
Ab6789, by Abcam (129 mentions)
Ab8227, by Abcam (126 mentions)
Ripa lysis buffer, by Beyotime (99 mentions)
Ab8245, by Abcam (90 mentions)
Ab181602, by Abcam (86 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (86 mentions)
Ab6728, by Abcam (75 mentions)
Ab32503, by Abcam (67 mentions)
Bca protein assay kit, by Beyotime (53 mentions)
Ab205719, by Abcam (52 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (48 mentions)
Bca kit, by Thermo Fisher Scientific (48 mentions)
Ab15580, by Abcam (44 mentions)
Ab8226, by Abcam (41 mentions)
Ab32124, by Abcam (38 mentions)
Ripa buffer, by Beyotime (37 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (37 mentions)
Ripa buffer, by Thermo Fisher Scientific (31 mentions)
1 526 protocols using ab6721
1
Western Blot Analysis of PRDX6 Protein
2
Podoplanin and VEGFR-3 Expression Analysis
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Proteins were extracted using radioimmunoprecipitation assay buffer and the protein concentration was established using the Bradford Protein Assay Kit (Bio Basic). Protein samples were diluted in 4X Laemmli buffer, then heated at 95°C for 5 minutes. Proteins were separated by electrophoresis on a 12% SDS‐PAGE, then transferred on nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris‐buffered saline (TBST, 0.1% Tween 20) for 1 hour at room temperature, then incubated with an anti‐podoplanin (OriGene, DP3512S), an anti‐vascular endothelial growth factor receptor (VEGFR)‐3 (Abcam, AB27278), or an anti‐beta‐actin (Abcam, AB8227) overnight at 4°C. The membranes were washed with TBST and incubated with horseradish peroxidase–conjugated secondary antibodies (Abcam, AB6721 and AB6721) for 1 hour at room temperature. Western Lightning Ultra chemiluminescence kit (PerkinElmer) was used for detection. Each sample was normalized with its respective beta‐actin expression.
3
Western Blot Analysis of Mesenchymal and Epithelial Markers
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MVs and cells were cleaved in radioimmunoprecipitation assay (RIPA) cell lysis reagent containing the protease inhibitor. The equivalent protein samples (15 μg) were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, and then mounted on polyvinylidene fluoride (Bio-Rad Laboratories, Inc., USA). After SDS-PAGE, the membrane was blocked with 5% skimmed milk at room temperature for 1 h and then incubated overnight with the primary antibodies at 4 °C. The primary antibodies were rabbit anti-human antibodies: anti-α-SMA antibody (ab5694, Abcam, UK), anti-FAP antibody (ab207178, Abcam, UK), anti-CEA antibody (ab207718, Abcam, UK), anti-CK-18 antibody (ab133263, Abcam, UK), anti-CD63 antibody (ab134045, Abcam, UK), anti-E-cadherin antibody (ab40772, Abcam, UK), N-cadherin antibody (ab76011, Abcam, UK), anti-vimentin antibody (ab92547, Abcam, UK), anti-SORBS1 antibody (ab224129, Abcam, UK), and anti-GAPDH antibody (ab6721, Abcam, UK). The secondary antibody was horseradish peroxidase-labeled goat antirabbit IgG antibody (ab6721, Abcam, UK).
4
Immunohistochemical Analysis of ELF3 and CLDN4
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The tissue samples were fixed, embedded in paraffin, cut into sections (4-μm thick), and then dewaxed, rehydrated, and soaked in 0.3% H2O2 to block the activity of endogenous peroxidase. After heat-mediated antigen retrieval in Tris/ethylene diamine tetraacetic acid buffer (pH = 9.0), the sections were blocked with 10% normal goat serum (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) and then incubated with the primary antibodies to ELF3 (1:250, ab97310, Abcam Inc., Cambridge, MA, USA) and CLDN4 (1:1000, ab6721, Abcam). After that, the sections were incubated with biotinylatedimmunoglobulin G (IgG, 1:1000, ab6721, Abcam). Then, the sections were stained with 3,3′-diaminobenzidine (DAB) and imaged under a microscope (OlympusOptical Co., Ltd., Tokyo, Japan) at a ×400 magnification.
5
Western Blot Analysis of Pancreatic Cancer Cells
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Panc1-WT and -KO and MiaPaCa2-WT and -KO protein lysates were prepared at p5; total protein lysates were obtained using 1x Laemmli buffer (BioRad, 1,610,737) at 4 °C under gentle agitation, followed by repeated sonication and collection of lysate by centrifugation. Protein levels were assessed by absorbance at 280 nm with nucleic acid correction, using NanoDrop™ One Spectrophotometer, (Fisher Scientific, 13–400-518). Equivalent (10 μg) protein was loaded onto 4–15% Tris HCL Protein Gels (BioRad, 4,561,086). Immuno-Blot PVDF membranes were used for transfer (BioRad, 1,620,174). Blots were blocked with 5% BSA for 2-h at 4 °C. Anti-GAPDH (Abcam, ab9485, 1:2500) and anti-β-actin (Abcam, ab8227, 1:2500) were used as protein loading controls. Protein levels were assessed using hu-6g8 (25μg/ml), anti-ADAR1 (1:2000), anti-Mcl-1 (Santa Cruz, sc-12,756, 1:200), anti-human IgG HRP (Sigma AP112P, 1:10000), anti-rabbit IgG HRP (Abcam, ab6721, 1:10000), and anti-mouse IgG HRP (Abcam, ab6721, 1:10000). Antibodies were incubated for 14 h at 4 °C. Blots were developed using SuperSignal West Pico Plus chemiluminescent substrate (Fisher Scientific, 34,580), and ImageQuant LAS 4000 biomolecular imager (GE Healthcare, Chicago, IL). Densometry analysis was performed in ImageJ software, with normalization to protein-loading controls.
6
Western Blot Analysis of Protein Expression
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Cells were lysed by Radio-Immunoprecipitation assay cell lysis buffer on ice and then centrifuged at 14,000 rpm for 20 minutes with the lysis buffer discarded to collect total protein. The protein concentration was determined using a bicinchoninic acid kit (Pierce, Rockford, IL, USA). Next, 30-50 μg total protein underwent 10% SDS-PAGE and transferred onto PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked in 5% skimmed milk for an hour and then incubated with the primary antibodies at 4℃ overnight and with the secondary antibody at 20℃ for an hour. The next day, the membranes were cultured with horseradish peroxidase-labeled secondary goat anti-rabbit antibody (1:2000, ab6721, Abcam) for 1 hour. Then the immunoreactive protein level was determined using an enhanced chemiluminescence kit (NEN, Dreieich, Germany). The antibodies used were against large tumor suppressor 2 (LATS2, 1:100, ab110780, Abcam), p-Yes-associated protein (p-YAP, 1:1000, Ser127, Cell Signaling Technology, MA, USA), YAP (1:500, 13584-1-AP, Proteintech, Rosemont, USA), β-actin (1:2500, AF5003, Beyotime) and horseradish peroxidase-labeled secondary antibody (1:3000, ab6721, Abcam).
7
Immunohistochemical Analysis of Metabolic Enzymes
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The aforementioned paraffin samples were sectioned and immersed in 3% methanol H2O2. Next, the sections were retrieved in antigen retrieval solution, followed by sealing in normal goat serum blocking solution at room temperature for 20 min. Subsequently, the sections were probed with the primary anti-rabbit polyclonal anti-GLS antibody (ab260047, dilution ratio of 1: 200, Abcam, Cambridge, UK), GDH (ab170895, dilution ratio of 1: 100, Abcam), RUNX3 (ab224642, dilution ratio of 1: 1000, Abcam) and FBXO4 (ab230302, dilution ratio of 1:1000, Abcam) at 4 °C overnight. The following day, the sections were incubated with the secondary antibody IgG (goat anti-rabbit, ab6721, dilution ratio of 1: 1000, Abcam) at 37 °C for 20 min. Afterwards, the sections were stained with DAB (ST033, Weijia Technology) development, and then counter-stained with hematoxylin. Thereafter, the sections were visualized under a microscope (CX43, Olympus Optical Co., Ltd., Tokyo, Japan). The Nikon image analysis software was utilized to document the positive cells. A total of 5 non-repetitive visual fields of equal area (200 times) were selected from each section and the number of positive cells and their proportions were calculated, with the average value calculated.
8
Immunohistochemical Analysis of KI67 Expression
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The tissues were fixed, dehydrated, and paraffin-embedded in the 10% formalin to make tissue sections of about 5 μm. The sections were dewaxed twice with xylene (15 minutes each time), followed by incubation in 3% H2O2 (Sigma-Aldrich) at 37°C for 30 minutes and boiling in 0.01 M citric acid buffer at 95°C for 20 minutes. After being blocked with serum working solution at 37°C for 10 minutes, the sections were incubated with diluted primary antibody to KI67 (1 : 1000, ab6721, Abcam) at 37°C for 2 h and with HRP-conjugated secondary immunoglobulin G (IgG) antibody and counterstained with hematoxylin (Meilunbio, China) at room temperature for 4 minutes. The sections were observed using a microscope (Olympus).
9
Immunohistochemical Analysis of Mammary Tissue
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IHC was performed by the Immunohistochemistry Core Laboratory at Texas A&M or the Pathology Core Laboratory at Baylor College of Medicine using standard protocols. Briefly, mammary sections from 6-month-old E-P72 and E-R72 FFPE mice were deparaffinized in xylene (VWR, MK866802), rehydrated in graded ethanols (100%–95%–70%) and distilled water. Antigen retrieval was performed in 0.1 M Tris-HCl, pH 9.0 for 15 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (VWR, BDH7540-2). Sections were blocked in 10% bovine serum albumin in Phosphate Buffered Saline with Tween 20 (PBST). Tissue sections were incubated for 45 min with primary antibody, and 1 h with HRP-conjugated anti-rabbit IgG secondary antibody (1:500, Abcam, ab6721). Color was developed by adding the substrate and chromogen 3,3′ -diaminobenzidine (DAKO). The primary antibodies used in IHC staining were Ki67 (1:500, Abcam, ab15580), CC3 (1:200, CST, 9664), IBA1 (1:500, WAKO, 019–19741), and CD31 (1:200, Abcam, ab28364). Sections were counterstained with hematoxylin (VWR, RC353032) and visualized with an automated upright microscope (LeicaDM5500B, Leica Biosystem, Germany). Quantification of images was performed using the ImmunoRatio plug-in, Fiji software [76 (link)] or blinded, manual counting of at least six high power fields.
10
Western Blot Analysis of TGFβ1 Signaling
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Cells were lysed with an RIPA lysis buffer (Solar life science, Beijing, China). Protein concentration was quantified by BCA Assay kit (Solar life science, Beijing, China). Proteins (100 μg) for each cell lysate sample were separated by 10% SDS-PAGE, which were then shifted to PVDF membrane (Bio-Rad, Hercules, CA). 5% BSA solution was used to block the membrane for 1h in room temperature. The primary antibodies were as follows: rabbit antibodies against TGFβ1 (1:1000; ab92486), Smad2 (1:800; ab63576), p-Smad2 (1:800; ab53100) and β-actin (1:1000; ab8227; Abcam, Cambridge, MA, UK) were incubated overnight at 4 °C. After washing in TBS containing Tween-20 (TBST) for 15 min, the membrane was incubated with HRP-labeled goat anti-rabbit (IgG-HRP, 1:1000; ab6721; Abcam, Cambridge, MA,UK) at room temperature for 2h. After washing in TBST for 15 min, chemiluminescent detection was performed using a Novex™ ECL Chemiluminescent Substrate Reagent kit (Thermo Fisher Scientific, Inc.). Protein expression level was detected using Bio-Rad Gel Doc XR + system (Bio-Rad, Hercules, CA, USA) and is presented as the density ratio versus β-actin.
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