For identification of the STBEV surface marker placental alkaline phosphatase (PLAP), western blotting was carried out on STBEV lysates. Briefly, 10ug/well per sample of total protein from normal STBEVs (n = 2) and PE STBEVs (n = 2) were separated on 4–20% bis-tris gradient gel (Bio-Rad, Hercules, CA, USA), using molecular weight standard (Precision Plus All Blue, Bio-Rad). Separated proteins were transferred onto PVDF membranes, and nonspecific binding blocked with 5% Blotting-Grade Blocker (Nonfat Dry Milk, Bio-Rad) in PBS-Tween. The membranes were incubated with the primary antibody NDOG2 against PLAP (made in-house and provided by Prof Sargent) overnight at 4 °C. Membranes were washed prior to incubation with the secondary antibody Alexa Fluor 647 goat anti-mouse IgG (Life Technologies, Carlsbad, CA, USA), washed again and thereafter the bands were detected in a ChemiDoc XRS unit (Bio-Rad).
Blotting grade blocker non fat dry milk
Manufactured by Bio-Rad
Sourced in United States
Blotting Grade Blocker Non-Fat Dry Milk is a laboratory product manufactured by Bio-Rad. It is used as a blocking agent in Western blotting and other immunoassay techniques to minimize non-specific binding of antibodies.
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Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (7 mentions)
Trans blot turbo transfer system, by Bio-Rad (5 mentions)
Chemidoc xrs imaging system, by Bio-Rad (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Chemidoc touch imaging system, by Bio-Rad (3 mentions)
Wet transfer apparatus, by Bio-Rad (3 mentions)
Edta free protease inhibitor, by Roche (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Mg132, by Merck Group (3 mentions)
Octet red96 system, by Molecular Devices (3 mentions)
Streptavidin coated biosensors, by Molecular Devices (3 mentions)
Hbs ep buffer, by GE Healthcare (3 mentions)
2 mercaptoethanol, by Bio-Rad (2 mentions)
Trans blot turbo transfer buffer, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
34 protocols using blotting grade blocker non fat dry milk
1
PLAP Expression in Placental STBEVs
2
Western Blot Protein Detection Protocol
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Cells were lysed in RIPA (Sigma) lysis buffer containing 1x Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher). Protein concentration was measured by Bio-Rad Bradford reagent. Protein samples were prepared by addition of 4x Laemmli Sample buffer (Bio-Rad) and 2-mercaptoethanol (Bio-Rad) and resolved on 4–12% SDS-PAGE (Sodium dodecyl sulfate–polyacrylamide) gels, which were subsequently transferred to PVDF membranes (Bio-Rad) using Trans-Blot Turbo transfer buffer (Bio-Rad) and a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% blotting-grade blocker non-fat dry milk (Bio-Rad), followed by overnight 4 °C incubation with the appropriate primary antibody and 1 h room temperature incubation with an anti-rabbit or anti-mouse IgG (H + L)-HRP conjugate (Bio-Rad) secondary antibody. Blots were imaged using Supersignal West Femto Maximum Sensitivity Substrate detection system (Thermo) and the ChemiDoc Imaging System (Bio-Rad). The following primary antibodies were used: c-Myc (Y69) (Abcam #ab32072, 1:1000 dilution), NME2 (4G7A8) (Abcam #ab204958, 1:1000 dilution), GAPDH (Cell Signaling Technology #3683, 1:5000 dilution), Actin (Cell Signaling Technology #5125 S, 1:5000 dilution).
3
Comprehensive Western Blotting Procedure
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Western blotting was carried out as previously described70 (link). Overall, lysates were prepared in RIPA buffer (Sigma) supplemented with protease (cOmplete Tablets, Mini, EDTA-free, Roche) and phosphatase (PhosSTOP, Roche) inhibitors. Protein lysates were loaded with 4× Laemmli sample buffer (Bio-Rad) and 2-mercaptoethanol (Bio-Rad) and run by SDS-PAGE, and were subsequently transferred to PVDF membranes (Bio-Rad) using Trans-Blot Turbo transfer buffer (Bio-Rad) and a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% blotting-grade blocker non-fat dry milk (Bio-Rad), followed by overnight 4 °C incubation with the appropriate primary antibody (Supplementary Table 3 ), and 1 h room temperature incubation with an anti-rabbit or anti-mouse IgG (H + L)-HRP conjugate (Bio-Rad) secondary antibody. Blots were imaged using chemiluminescent substrate (Thermo Scientific) and a LAS-3000 imager (Fujifilm) or ChemiDoc Imaging System (Bio-Rad). When necessary, blots were stripped with Restore PLUS western blot stripping buffer (Thermo Scientific), followed by repeat blocking and antibody incubations. Uncropped images of the western blots from Fig. 5a are presented in Supplementary Figure 14 .
4
Western Blot and ELISA Protocols
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Equal amounts of each sample (25-50 µg) were electrophoresed on sodium dodecyl sulfate–polyacrylamide gels electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes. Membranes were pre-incubated with 5% blotting grade blocker non-fat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) in TBS with 0.1% Tween 20 (TBS-T) at room temperature for 1 h and blotted over night at 4°C with the specific primary antibodies. Antibody-specific labeling was revealed by incubation with a HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody (1:5000) and visualized with the ECL chemiluminescence kit (Amersham Biosciences). Cytokine production in vitro by macrophages and its accumulation in the culture medium was quantified using an ELISA Bioplex kit (Bio-Rad) according to the manufacturer’s instructions.
5
Western Blot Analysis of Key Stem Cell Markers
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Whole-cell lysates were generated by incubating cells in RIPA buffer for 30 min on ice, followed by sonication using a Covaris S-220 Ultrasonic Processor for 5 min. Lysates were separated in an 8% polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore) using an OWL semi-dry transfer apparatus. Membranes were blocked using 1% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad) and incubated overnight at 4°C with the following primary antibodies: a rabbit polyclonal RUNX1 (Cell Signaling #4334, 1:1,000); a goat polyclonal to POU5F1 (Santa Cruz Biotechnology #sc-8628, 1:1,000); a rabbit polyclonal to CDK2 (M2) (Santa Cruz #sc-163, 1:2,000); a mouse monoclonal to GAPDH (0411) (Santa Cruz #sc-47724); a rabbit monoclonal to SMAD2 (D43B4) (Cell Signaling #5339); and a rabbit monoclonal to pSMAD2 (Ser465/467) (138D4) (Cell Signaling #3108). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz) were used for immunodetection, along with the Clarity Western ECL Substrate (Bio-Rad) on a Chemidoc XRS+ imaging system (Bio-Rad).
6
Characterization of EGFR+ HNSCC Tumor Cells
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The murine EGFR+ HNSCC tumor cell line MOC2, established from a carcinogen-induced oral SCC in a C57BL/6 mouse (30 (link)), was purchased from Kerafast, Inc. The cell line was authenticated by STR (short tandem repeat) and was free of pathogens. Purified fusion toxins were analyzed using 4–12% SDS-PAGE gels. The gels were stained with Gel Code Blue Staining Reagent (Thermo Fisher Scientific, Inc.) and mounted with DryEase Mini Cellophane (Thermo Fisher Scientific, Inc.). Western blot analysis was performed as previously described (31 (link)). Briefly, proteins (~1 µg in 15 µl per sample) were separated on 4–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Thermo Fisher Scientific, Inc.). The membranes were blocked with 5% blotting grade blocker non-fat dry milk (Bio-Rad Laboratories, Inc.) in 1X PBS, 0.02% Tween 20 for 1 h with shaking and washed once with 1X PBS, pH 7.4, 0.2% Tween 20 at room temperature with shaking. The three EGF fusion toxins were detected using mouse anti-DT, mouse anti-human EGF, or mouse anti-human IL2 primary antibodies (1:1,000) and rat anti-mouse IgG-HRP as the secondary antibody (1:4,000). The proteins were detected using the TMB (Tetramethylbenzidine) membrane peroxidase substrate (SeraCare; LGC Clinical diagnostics). The antibodies used in the present study are listed in Table SI .
7
Western Blot Analysis of MSMB and Ubiquitin
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The nitrocellulose membranes with transferred proteins were blocked for one hour with 5% non-fat milk (Blotting Grade Blocker Non-Fat Dry Milk, Bio-Rad) in PBS-Tween (0.5% Tween 20; Sigma-Aldrich) and incubated in parallel with primary antibodies anti-MSMB (1:500 dilution, polyclonal rabbit antibody) and anti-ubiquitin antibody (FK2, 1:250 dilution, monoclonal mouse antibody recognizing mono- and polyubiquitinated conjugates; ENZO Life Sciences), both in 1% non-fat milk in PBS-Tween, overnight. For protein normalization purposes, the membranes were stripped and incubated with monoclonal antibody anti-alpha-tubulin DM1A (1:5000 dilution; Sigma-Aldrich). The following day, membranes were washed in PBS-Tween and incubated with HRP-conjugated species-specific secondary antibodies such as goat anti-rabbit IgG and goat anti-mouse IgG (1:3000 dilution; Bio-Rad) in 1% non-fat milk in PBS-Tween for 60 min at laboratory temperature. The membranes were washed four times in PBS-Tween and two times in PBS, reacted with a chemiluminescent substrate (Super Signal West Pico Chemiluminescent Substrate; ThermoFisher Scientific), and reactive bands were screened with Azure c600 imaging system (Azure Biosystems).
8
Western Blot Protein Analysis
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Cells were lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with 100× protease and phosphatase inhibitor (Thermo Fisher Scientific) and sonicated. Samples were resolved using 4–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad) at a constant voltage. The membrane was blocked using either 5% (v/v) BSA in Tris-buffered saline with 0.1% Tween-20 (TBST) or 5% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad) prior to incubation with antibodies of interest. Proteins were visualised using SuperSignal® West Dura Extended Duration Substrate (Thermo Fisher Scientific) and imaged using ChemiDoc MP Imaging System (Bio-Rad).
9
Protein Extraction and Quantification from Spheroids
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Spheroids were digested in 100 µL RIPA Buffer (Sigma-Aldrich); then, protein lysates were collected, mixed with ×1 Halt proteinase inhibitor cocktail mix (Thermo Scientific), and sonicated twice in 3-s intervals at 50% power. Sample concentrations were determined via Pierce BCA Protein Assay (Thermo Scientific), and then samples containing ×4 Laemmli Sample Buffer (Bio-Rad), 2 Mercaptoethanol (Bio-Rad), and 7.5 µg protein were run on an Expressplus PAGE 4–20% Gel (GenScript) at 200 V for 30 min. Proteins were transferred to nitrocellulose membranes with a Trans-Blot Turbo Transfer System (Bio-Rad); then, the membranes were blocked in 5% Blotting Grade Blocker Non Fat Dry Milk (Bio-Rad) for 30 min and stained with primary and secondary antibodies (Supplemental Table S1) for 1 h each. Blots were incubated for 5 min in Pierce SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and imaged with the ChemiDoc Touch Imaging System (Biorad). Protein bands were quantified with Image Lab Software (Bio-Rad); results for each sample were normalized to intrinsic beta actin abundance and to measurements made on the first day after spheroid assembly for each batch of spheroids. Data was collected from four independent batches of spheroids for each protein.
10
Protein Extraction and Western Blot Analysis
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Protein samples were extracted with TRIzol reagent (Invitrogen) and were dissolved in an amphoteric electrolyte. Western blot assays were performed as described previously (Huang et al., 2012 ). Nitrocellulose membranes were blocked using 5% Blotting Grade Blocker Non‐Fat Dry Milk (Bio‐Rad, Hercules, CA, USA) and were then incubated with primary antibodies at 4 °C overnight. For primary antibodies, anti‐CD26 was purchased from R&D Systems (Minneapolis, MN, USA); anti‐CD44, anti‐p65, anti‐p‐p65, anti‐SETD7 anti‐VDR and anti‐GAPDH were purchased from Cell Signaling Technology (CST, Danvers, MA, USA); and anti‐SPOP was purchased from Proteintech (Wuhan, China). The blots were then incubated with horseradish peroxidase‐conjugated secondary antibody (CST) at room temperature for 1 h. Bands were analyzed by gel‐pro analyzer software (Media Cybernetics).
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