Western blotting was performed according to the standard protocols and probed with PAI-1 (R&D Systems, MAB1786), TFPI (Abcam, ab180619), PAR-1 (Sigma, sab4500823), VCAM1 (Novus, NBP2-29413), ICAM1 (Novus, NBP2-67518), and GAPDH (Santa Cruz Biotechnology, SC365062). Blots were imaged using a Konica Minolta SRX-101A Medical film processor and quantified with ImageJ software. The scanned Western blot images were imported into the ImageJ software. Regions of interest (ROIs) were defined around the bands corresponding to the protein of interest and the reference protein, GAPDH, by selecting the “rectangle tool.” Densitometric analysis was then conducted with the ImageJ using the “measure” option to quantify the intensities of these bands. The obtained target protein intensities were then normalized to the corresponding GAPDH bands, which served as an internal control.
Sc 365062
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom, China
Sc-365062 is a laboratory equipment product from Santa Cruz Biotechnology. It is designed for general laboratory use, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Anti gapdh, by Santa Cruz Biotechnology (9 mentions)
Gapdh, by Santa Cruz Biotechnology (8 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Na934v, by GE Healthcare (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
P0260, by Agilent Technologies (3 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ab103328, by Abcam (2 mentions)
A11012, by Thermo Fisher Scientific (2 mentions)
Na931v, by GE Healthcare (2 mentions)
Sc 376382, by Santa Cruz Biotechnology (2 mentions)
A11001, by Thermo Fisher Scientific (2 mentions)
Mabe343, by Merck Group (2 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
76 protocols using sc 365062
1
Western Blot Protein Expression Analysis
2
Western Blot Protein Analysis
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After electrophoresis, the proteins were blotted into polyvinylidene difluoride membranes under semi-dry transfer conditions (Thermo Scientific, Rockford, IL). After blocking with 5% milk, the following antibodies were used: anti-VDR (1:200, sc-13133; Santa Cruz Biotechnology, Dallas, TX), anti-TNF-α (1:500, ab183896; Abcam, Cambridge, United Kingdom), and secondary antibodies (Code: 115-035-146; Code: 111-035-003, Jackson ImmunoResearch, West Grove, PA). Protein loading was normalized to anti-GAPDH (1:5,000, sc-365062; Santa Cruz). Bands were visualized by chemiluminescence detection (Chemiluminescent HRP Substrate, Millipore, MA) and quantified using the MicroChemi 2.0 System. Specific bands were quantified by densitometric analyses with GelQuant software (Jerusalem, Israel).
3
Western Blot Analysis of TGFBR2 Protein
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Cells or tissues were lysed in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate and 1 mM EDTA, pH 8.0) containing freshly added protease inhibitor cocktail (Roche) for 30 min on ice and were then centrifuged at 16,000 × g at 4°C for 10 min. The supernatant was collected, and the protein concentration was calculated using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The proteins were separated via SDS-PAGE (Bio-Rad). After electrophoresis, the proteins were electrotransferred to PVDF membranes (Bio-Rad) and then blocked with 5% skim milk for 1 h. The membranes were then incubated in primary antibodies at 4°C for 12 h. After three washes in TBST, the membranes were incubated in a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After three washes, the membranes were incubated in the SuperSignal West Pico chemiluminescence substrate (Pierce). The same membrane was probed with a GAPDH antibody to serve as a loading control. The antibodies against TGFBR2 and GAPDH were purchased from Santa Cruz Biotechnology (sc-400 and sc-365062, respectively; Santa Cruz, CA, USA).
4
Western Blot Analysis of Cell Proteins
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Total protein from cells was extracted in lysis buffer (Pierce) and quantified using the Bradford method. Then, proteins (50 µg) were separated by SDS-PAGE (12%) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Subsequently, membranes were incubated overnight at 4°C with monoclonal antibodies against E-cadherin (sc-8426, 1:200; Santa Cruz Biotechnology), β-catenin (610154, 1:500; BD Transduction Laboratories, Lexington, KY, USA) or GAPDH (sc-365062, 1:500, Santa Cruz Biotechnology). After incubation with peroxidase coupled anti-mouse IgG (Santa Cruz Biotechnology) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce) and detected using BioImaging Systems (UVP Inc., Upland, CA, USA). The relative protein levels were calculated based on GAPDH protein as a loading control.
5
Immunofluorescence and Immunoblotting Antibodies
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The following antibodies were used for immunofluorescence experiments: rabbit anti-TIAR (5137S, Cell Signaling Technology, Lot #1, 1:100), mouse anti-TIAR (Clone 6) (610352, BD Biosciences, Lots #5357680; 7219778, 1:100), mouse anti-Edc4 (H-12) (sc-376382, Santa Cruz Biotechnology, Lot #I0216, 1:100), mouse anti-Puromycin clone 12D10 (MABE343, Millipore Sigma, Lot #2861354, 1:100), rabbit anti-G3BP1 (13057–2-AP, Proteintech, Lot #00047654, 1:100), mouse anti-ATXN2 (Clone 22) (611378, BD Biosciences, Lot #7341666, 1:100), rabbit anti-YTHDF1 (17479-AP, Proteintech, Lot #00040713, 1:100), rabbit anti-YTHDF2 (24744–1-AP, Proteintech, Lot #00053880, 1:100), rabbit anti-YTHDF3 (ab103328, Abcam, Lot #GR35115–39, 1:100), rabbit anti-IgG Alexa Fluor 594 (A11012, Invitrogen, Lot #1933366, 1:1000), mouse anti-IgG Alexa Fluor 488 (A11001, Invitrogen, Lot #1939600, 1:1000).
The following antibodies were used for immunoblotting experiments: rabbit anti-YTHDF2 (ARP67917_P050, Aviva System Biology, Lot #QC38405–43182, 1:1000), mouse anti-GAPDH (SC-365062, Santa Cruz Biotechnology, Lot #A2816, 1:5000), rabbit anti-IgG HRP (NA934V, GE Healthcare, Lot #16677077, 1:10000), mouse anti-IgG HRP (NA931V, GE Healthcare, Lot #16814909, 1:10000).
The following antibodies were used for immunoblotting experiments: rabbit anti-YTHDF2 (ARP67917_P050, Aviva System Biology, Lot #QC38405–43182, 1:1000), mouse anti-GAPDH (SC-365062, Santa Cruz Biotechnology, Lot #A2816, 1:5000), rabbit anti-IgG HRP (NA934V, GE Healthcare, Lot #16677077, 1:10000), mouse anti-IgG HRP (NA931V, GE Healthcare, Lot #16814909, 1:10000).
6
SOCS3 Protein Expression in PAH
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Western blot analysis was performed using frozen PBMCs from N = 12 PAH patients vs. N = 5 CTRLs. The membranes were incubated with both mouse anti-human anti-SOCS3 (OriGene, #TA502991) at a dilution 1:1000 and mouse anti-human anti-GAPDH at a dilution 1:5000 (Santa-Cruz #sc-365062) overnight at 4 °C and next incubated with peroxidase-labeled secondary antibody and visualized using the ECL detection system (Data Supplement). GAPDH protein served as loading control and was detected on the same membranes for normalizing the signals.
7
Protein Extraction and Western Blot Analysis
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We extracted protein from cells or tissues with RIPA lysis buffer. Western blot assays were performed following a previous procedure [18 (link)]. We separated protein samples (20 μg) using a 10% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel and transferred the proteins to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA). We blocked membranes with nonfat dry milk (Yili Milk Company, Inner Mongolia, China) at room temperature for 2 hours, and then hybridized with anti-HOXC9 (1:1000 dilution; SC-81100; Santa Cruz Biotechnology, CA.USA) and anti-GAPDH antibodies (1:2000 dilution; SC-365062; Santa Cruz Biotechnology) at 4°C overnight. We next added secondary biotin-conjugated antibodies, and developed immunoreactive bands with the enhanced chemiluminescence kit (Thermo Fisher Scientific). We used GAPDH as a loading control.
8
Western Blot Analysis of Protein Samples
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Samples were prepared with reducing Laemmli buffer, denatured by heating at 95 °C for 10 min and subjected to SDS-PAGE. By semi-dry blotting, proteins were transferred onto a nitrocellulose membrane (#10600002, GE Healthcare Life Sciences, Uppsala, Sweden) that was then blocked with 5% nonfat dry milk in PBS-T (PBS containing 0.1% Tween-20) for 1 h at room temperature. Primary antibodies were diluted in PBS-T as well and incubated accordingly. Antibodies used were directed against His6 (1:1.000, #MA1-21315, Invitrogen, Carlsbad, CA, USA), Hsp90 (1:1.000, #sc-13119, SantaCruz Biotechnology, Dallas, TX, USA) and GAPDH (1:1.000, #sc-365062, SantaCruz Biotechnology, Dallas, TX, USA), all incubated 1 h at room temperature. Anti-NDPK-A (1:200, #sc514515, SantaCruz Biotechnology, Dallas, TX, USA) was applied over night at 4 °C. For detection an anti-mouse IgG kappa binding protein (m-IgGκ BP) HRP-conjugate (1:2.500, #sc-516102, SantaCruz Biotechnology, Dallas, TX, USA) was used, diluted in PBS-T and incubated for 1 h at room temperature. For visualization, the ECL system (#WBKLS0500, EMD Millipore Corporation, Darmstadt, Germany) was used.
9
Western Blot Analysis of PARP1 Protein
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Samples of an identical amount of protein (5 μg) were separated using SDS-PAGE electrophoresis according to Laemmli [31 (link)] and were then transferred to nitrocellulose sheets, according to the Towbin et al. protocol [31 (link),32 (link)]. The quality of transfer was determined with Ponceau S staining. Then, the membranes were blocked with 5% non-fat milk in Tris-Buffered Saline with Tween 20 (TBST) for 1 h at RT and incubated overnight at 4 °C with primary mouse antibodies directed against PARP1 (SC-74470; dilution 1:500; clone B-10, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-365062, Santa Cruz Biotechnology, Santa Cruz, CA, USA), which was used as an internal loading control. This was followed by a 1 h incubation with secondary antibodies conjugated with horseradish peroxidase directed against primary mouse antibodies (Cell Signaling, Danvers, MA, USA). Immunoblots were developed using the Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA), scanned with ChemiDoc (Bio-Rad, USA) and were analyzed with ImageLab software (ver. 6.0, Bio-Rad, USA). The experiment was performed in triplicate (three biological repetitions, each consisting of two–three technical replicates).
10
Western Blot Analysis of Epithelial and Mesenchymal Markers
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Cells grown in culture dishes were trypsinized and centrifuged at 1,500 rpm for 3 min. Cell pellets were lysed with Radio-Immunoprecipitation Assay buffer and sonicated. The lysates were centrifuged at 9300xg for 5 min to remove debris. The protein concentration in the supernatant was determined with the Pierce BCA protein assay kit (23225, ThermoFisher Scientific, United States). 30–50 µg protein was boiled for 10 min with the addition of 4X Laemmli buffer. The boiled lysates were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were blocked with 5% non-fat dry milk in 1X TBST before incubating overnight with respective antibodies–vimentin (1:1,000, CPCA-Vim, EnCor Biotechnology), Keratin8 (1:1,000, 1,432-1, Epitomics, United States) and GAPDH (1:1,000, sc-365062, Santa Cruz Biotechnology, United States). After washing the blots three times for 5 min each with 1X TBST, they were incubated for 1 h with fluorescent secondary antibodies in 1X TBST (1:10,000, IRDye 800CW, 680RD, LI-COR Biosciences, United States). The blots were then imaged with an Odyssey Fc Imaging System (LI-COR Biosciences, United States) after washing for 3X (5 min) with 1X TBST.
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