CagA protein and exosome markers (CD9 and HSP70) were detected by western blotting. Equal amounts of cell lysates and exosomes were separated by 7.5% or 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (ATTO Co., Ltd., Japan). After blocking with 0.5% skim milk or Blocking One-P (Nacalai Tesque Inc., Kyoto, Japan) in TBST (20 mM Tris, 500 mM NaCl, pH 7.4, 0.05% Tween 20), membranes were blotted with the following primary antibodies: anti-HA antibodies (3F10; Roche), anti-CagA antibodies (AUSTRAL Biologicals), anti-phosphotyrosine antibodies (ab10321 [PY20]; Abcam), anti-CD9 antibodies (ab92726; Abcam), and anti-HSP70 antibodies (ab5439; Abcam). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and ECL Western Blotting Detection Reagents (GE Healthcare). The bands were visualised using LAS-4000 (GE-Healthcare). For detection of tyrosine-phosphorylated CagA, the blot was first probed with anti-phosphotyrosine antibodies, stripped with EzReprobe reagent (ATTO Co., Ltd., Japan), and then reprobed with anti-CagA antibodies.
Ab5439
Manufactured by Abcam
Sourced in United Kingdom
Ab5439 is a primary antibody that recognizes the CD133 protein. CD133 is a cell surface glycoprotein that is commonly used as a marker for stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Ab92726, by Abcam (3 mentions)
Mab1501, by Merck Group (2 mentions)
Bca kit, by Nanjing Jiancheng (2 mentions)
Las4000, by GE Healthcare (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Blocking one p, by Nacalai Tesque (1 mentions)
Anti ha antibodies 3f10, by Roche (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Caga sc 28368, by Santa Cruz Biotechnology (1 mentions)
0.22 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab69659, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti mouse igg or anti rabbit igg, by Abcam (1 mentions)
Ab79868, by Abcam (1 mentions)
Ab13492, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Dab horseradish peroxidase color development kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Sp 9002, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
13 protocols using ab5439
1
Western Blot Analysis of CagA and Exosome Markers
2
Western Blot Protein Expression Analysis
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The Western blot was used to determine the expression of all target proteins, and the process was performed in accordance with a previous study [75 (link)]. The primary antibodies used in present study were as follows: GRP78 (1:1000), GPX1 (1:1000), GPX4 (1:2000), and TXNRD2 (1:1000) (200310-4F11, 616958, 513309, R26013; Zen BioScience, Chengdu, China); HSP70 (1:5000) (ab5439; Abcam, Cambridge, UK); SELENOS (1:1000) (15591-1-AP, ProteinTech Group, Chicago, IL, USA); and β-Actin (1:5000; MAB1501; Millipore, Darmstadt, Germany).
3
Western Blot Analysis of CagA, HSP70, and CD9
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The levels of CagA protein and exosome markers (HSP70 and CD9) were determined using Western blotting. The protein preparations (30 μg proteins) were loaded on 5% and 10% SDS‐PAGE. After electrophoresis, the preparations were transferred to polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA). After blocking with 5% skim milk in tris‐buffered saline with Tween for 1 hour, the membranes were incubated with the following primary antibodies: CagA (sc‐28368; diluted factor 1:200, Santa Cruz, Dallas, TX), Hsp70 (ab5439; dilution factor 1:1000, Abcam, Cambridge, MA), CD9 (ab92726; dilution factor 1:1000, Abcam, Cambridge, MA) at 4°C overnight. Then membranes were incubated with IRDye secondary antibodies (LI‐COR, dilution factor 1:10 000, Li‐Cor Biosciences, Lincoln, NE). The membranes were visualized using Odyssey CLx Imaging System (Li‐Cor Biosciences, Lincoln, NE).
4
PRRSV Infection and PAM Protein Analysis
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Samples of PRRSV‐infected and DMEM‐inoculated PAMs were lysed at 24 h post infection and protein concentrations were determined. Samples (20 μg) were separated by 12% SDS‐PAGE and transferred to 0.22 μm nitrocellulose membranes (Bio‐Rad Laboratories, Hercules, CA, USA). Membranes were blocked with 5% skim milk in Tris‐buffered saline containing 0.05% Tween‐20 and incubated overnight at 4 °C with monoclonal antibodies against heat shock protein 70 (HSP70; ab5439; Abcam plc, Cambridge, UK) or KDEL receptor (ab69659; Abcam plc). After washing three times, membranes were incubated at 37 °C for 60 min with horseradish peroxidase‐conjugated anti‐mouse IgG or anti‐rabbit IgG (Abcam plc). Detection used chemiluminescence luminal reagents (Pierce Biotechnology, Waltham, MA, USA).
5
Immunohistochemical Analysis of Heat Shock Proteins
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Immunohistochemistry was performed as previously described [43 (link), 44 (link)]. The paraffin sections (5 μm) were deparaffinized in xylene, rehydrated with a graded series of ethanol, and washed in water. Antigen retrieval was performed by microwaving the sections in 10 mM sodium citrate buffer (pH 6.0) followed by cooling the sections to room temperature. The endogenous horse radish peroxidase activity was inhibited with 3% H2O2 for 15 min. After nonspecific binding was blocked with 10% horse serum at 37 °C for 1 h, the sections were incubated with primary antibody at 4 °C overnight. The primary antibodies used in this study include mouse anti-HSP27 antibody (1:800; ab79868, Abcam, Cambridge, UK), mouse anti-HSP70 antibody (1:800; ab5439, Abcam) and mouse anti-HSP90 antibody (1:400; ab13492, Abcam). The sections were then incubated with a biotin-labeled goat anti-mouse IgG antibody (1:1000; ab6789, Abcam) and a streptavidin-conjugated HRP complex (SP9002, Zhongshan Golden Bridge, Beijing, P. R. China). Finally, the signals were visualized with the DAB Horseradish Peroxidase Color Development Kit (ZLI9018, Zhongshan Golden Bridge) and counterstained with hematoxylin.
6
EV Protein Expression Analysis
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Anti-CD9 rabbit monoclonal antibody (ab92726; Abcam, Cambridge, UK), anti-HSP70 mouse monoclonal antibody (ab5439; Abcam), anti-CD81 mouse monoclonal antibody (sc-166029; Santa Cruz Biotechnology, CA, USA), horse-radish peroxidase (HRP)-conjugated anti-rabbit IgG polyclonal antibody (A24531; Thermo Fisher Scientific, Waltham, MA, USA), and HRP-conjugated anti-mouse IgG rabbit polyclonal antibody (ab97046; Abcam) were purchased as indicated. To observe the expression of CD9, HSP70, and CD81 in the collected EVs, the EVs (1 μg protein) were subjected to 10% (for CD9 and HSP70) or 12.5% (for CD81) SDS-PAGE, and the proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The PVDF membrane was incubated with 3% bovine serum albumin (BSA) dissolved in Tris-HCl-buffered saline containing 0.1% Tween20 (pH 7.4) for 1 h at 37 °C, then with anti-CD9 antibody (1:2000), anti-HSP70 antibody (1:2000), or anti-CD81 antibody (1:100) for 24 h at 4 °C, respectively. Thereafter, the membrane was incubated with each secondary antibody at a dilution of 1:2000 for rabbit IgG or 1:5000 for mouse IgG for 1 h at 37 °C. After incubation of the membrane with a chemiluminescent substrate reagent (ECL prime; GE Healthcare, Little Chalfont, UK), the bands of each protein were detected with a LAS-4000 mini system (Fuji Film, Tokyo, Japan).
7
Quantification of Liver Protein Abundance
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Liver total protein extracts were prepared using RIPA lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.25% sodium deoxycholate, 1% NP-40, 1 mmol/L EDTA, 10 μL/mL protease inhibitor, 10 μL/mL phosphatase inhibitor 3, 10 μL/mL 100 μmol/L Na3VO4, 10 μL/mL 10 mg/mL PMSF], and measured the protein concentration using a BCA kit (Jiancheng Bioengineering, China). Fixed protein amounts were electrophoresed using 12% SDS-PAGE gel and blotted onto PVDF membrane (Millipore, USA). The membranes were blocked and immunoblotted with primary antibodies against target protein HSP70 (1:5,000; ab5439; Abcam), AMPKα (1:1,000; #5831; Cell Signaling Technology), p-AMPKα (1:1,000; #2535; Cell Signaling Technology), GPX1 (1:1,000; 616,958; Zen BioScience), GPX4 (1:2,000; 513,309, Zen BioScience), SELENOS (1:1,000, 15,591–1-AP, Proteintech Group) and β-ACTIN (1:5,000; MAB1501; Millipore), respectively. Then incubated with corresponding secondary antibodies (horseradish peroxidase-linked goat anti-rabbit or mouse IgG). Autoradiography and chemiluminescence with an enhanced chemiluminescence system (Millipore, USA) was applied to detect and quantify the signal. Image Lab™ software system (Bio-Rad, USA) was used to analyze the densitometric of western blot bands. The ratio of target protein to β-actin protein represented the relative abundance of each target protein.
8
Quantifying HSP70 and Annexin A2 in CTEPH
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Histological sections were produced with 3-mm thickness containing representative samples of endothelium extracted from the thrombus of patients with CTEPH, which was previously stored at -80°C. Transplanted lungs were used as control tissue by using pieces of the pulmonary artery of the donor. All the samples underwent immunohistochemical analysis for the identification of cytoplasmic HSP70 (Abcam, anti-Hsp70 antibody (3A3), #ab5439) and annexin 2 (Abcam, anti-annexin A2 antibody C-terminal, #ab185957), according to the manufacturer’s protocol. Histomorphometric quantification of the expression of the HSP70 and annexin A2 markers and total area quantification randomly of 5 different fields was performed by using a microscope-coupled image analyzer. The system consists of an Olympus-5 camera coupled to an Olympus microscope, from which the images are viewed on a monitor and evaluated on a digital imaging system (Software Image Pro-Plus 6.0).
9
Western Blot Analysis of Antioxidant Proteins
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The skeletal muscle tissues were homogenized with the cell disruption buffer (RIPA lysis Buffer, Beyotime, Shanghai, China), then the total protein concentration was measured using the BCA kit (Jiancheng Bioengineering, Nanjing, China). The subsequent Western blot process was performed as previously described [11 (link),29 (link)]. The primary antibodies were used at the following dilutions: HSP70 (1:5000; ab5439; Abcam, Cambridge, UK), GPX1 (1:1000; 616958; Zen BioScience, Chengdu, China), GPX3 (1:2000; sc-58361, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPX4 (1:2000; 513309, Zen BioScience, Chengdu, China), SELENOP (1:2000; sc-376858, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SELENOS (1:1000, 15591-1-AP, ProteinTech Group, Chicago, IL, USA) and GAPDH (1:5000; 200306-7E4, Zen BioScience, Chengdu, China).
10
Extracellular Vesicle Protein Profiling
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Samples were first applied to a 12% Tris-HCl gel and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were then blocked with 5% skim milk or 5% BSA in 0.05% Tween 20-TBS (Tris-buffered saline) (USB Corporation, Fremont, CA, USA) for 1 h and incubated with a primary antibody anti-CD63 (ab68418, Abcam, 1 : 1000), anti-CD81 (ab155760, Abcam, 1 : 1000), anti-Hsp70 (ab5439, Abcam, 1 : 1000), and anti-NFAM1 (bs-2680R, Bioss, 1 : 500) overnight at 4°C and then detected with a secondary antibody (7074S, Cell Signaling) for 1 h at room temperature. The protein bands were visualized with enhanced chemiluminescence and normalized to β-actin. Images were processed using ImageJ version 1.41 (National Institutes of Health, Bethesda, MD, USA).
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