PBS and Protease inhibitor cocktail, were purchased from Sigma Aldrich-Company (Saint-Quentin Fallavier, France). The 4%-12% Bis-Tris gels, Invitrolon PVDF membranes, Seeblue multi-colored standard were obtained from Invitrogen (Cergy Pontoise, France). The goat polyclonal antibody anti COX-2 (sc-1747), and the secondary antibodies IgG1 horseradish conjugated were obtained from Santa Cruz biotechnology (Tebu, Le Perray-en-Yvelines, France).
Invitrolon pvdf membrane
Manufactured by Thermo Fisher Scientific
Sourced in United States
Invitrolon PVDF membranes are a type of laboratory equipment used for various applications in the scientific and research community. These membranes are made of polyvinylidene fluoride (PVDF), a material known for its chemical and physical properties. The primary function of Invitrolon PVDF membranes is to facilitate the separation, transfer, and analysis of biomolecules, such as proteins and nucleic acids, during various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (4 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (4 mentions)
Nupage, by Thermo Fisher Scientific (3 mentions)
Nupage gel, by Thermo Fisher Scientific (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
N per, by Thermo Fisher Scientific (2 mentions)
Nupage 1.5mm 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Ir secondary antibody, by LI COR (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Proteinase and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Rabbit anti akt antibody, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase linked secondary antibody, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitors 2 and 3, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Supersignal west femto reagent, by Thermo Fisher Scientific (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
27 protocols using invitrolon pvdf membrane
1
COX-2 Protein Expression Analysis
2
Immunoblotting for Signaling Proteins
3
Protein Expression and Western Blot Analysis
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Cells were lysed
with RIPA buffer (Sigma)
supplemented with protease inhibitor (Roche) and phosphatase inhibitors
II and III (Sigma) and analyzed by SDS-PAGE with 4–12% Bis·Tris
gels (Nupage, Invitrogen), followed by electrotransfer onto Invitrolon
PVDF membranes (Invitrogen). Membranes were blocked with nonfat dry
milk (5% w/v) and Tween-20 (0.1% v/v) in tris-buffered saline (TBS-T),
for 1 h at room temperature before being probed with the primary antibody
by overnight incubation at 4 °C, followed by incubation
for 1 h at room temperature with a horseradish-peroxidase-linked secondary
antibody (Cell Signaling) and detection with the aid of Clarity Western
ECL Substrate (BioRad), by the manufacturer’s protocol. Immunoblots
were quantified by scanning the films and then performing densitometry
by use of ImageJ (US National Institutes of Health;http://rsb.info.nih.gov/ij/ ). Differences between three concentrations of 1 were
evaluated by the two-way analysis of variance (ANOVA) with Sidak’s
multiple comparisons test. Values represent means ± SEM determined
from the results of three independent experiments. A value of p < 0.05 was considered statistically significant.
with RIPA buffer (Sigma)
supplemented with protease inhibitor (Roche) and phosphatase inhibitors
II and III (Sigma) and analyzed by SDS-PAGE with 4–12% Bis·Tris
gels (Nupage, Invitrogen), followed by electrotransfer onto Invitrolon
PVDF membranes (Invitrogen). Membranes were blocked with nonfat dry
milk (5% w/v) and Tween-20 (0.1% v/v) in tris-buffered saline (TBS-T),
for 1 h at room temperature before being probed with the primary antibody
by overnight incubation at 4 °C, followed by incubation
for 1 h at room temperature with a horseradish-peroxidase-linked secondary
antibody (Cell Signaling) and detection with the aid of Clarity Western
ECL Substrate (BioRad), by the manufacturer’s protocol. Immunoblots
were quantified by scanning the films and then performing densitometry
by use of ImageJ (US National Institutes of Health;
evaluated by the two-way analysis of variance (ANOVA) with Sidak’s
multiple comparisons test. Values represent means ± SEM determined
from the results of three independent experiments. A value of p < 0.05 was considered statistically significant.
4
Western Blot Analysis of NBCe1 Protein
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Protein extracts were run on NuPAGE 3%–8% Tris-acetate gels alongside 5 µL of BLUelf prestained protein ladder (FroggaBio, Concord, ON, Canada) or HiMark prestained protein standard (Thermo Fisher Scientific). Blank lanes were loaded with 20 µL NuPAGE LDS sample buffer. Protein bands were transferred onto Invitrolon PVDF membranes (Invitrogen, Carlsbad, CA, USA) and stored in blocking buffer (5% milk powder and 0.1% Tween 20 in TBS) overnight. NBCe1 protein was probed using a 1:1000 dilution of rabbit anti-SLC4A4 polyclonal antibody (#E-AB-14348, Elabscience, Houston, TX, USA; see [38 (link)] and Supplemental Figure S2 for validation) or 1:1000 of our novel rabbit anti-pSer982 antibody (preabsorbed for 30 min with 16 µg/10 mL blocking buffer of peptide (Pep1); see manuscript results for details of validation). Exceptions were the data in Figure 9 , for which we incidentally used a different anti-NBCe1-A/B antibody [39 (link)] at a 1:1000 dilution. Immunoreactivity was disclosed using 1:2000 of HRP-conjugated goat-anti-rabbit secondary antibody (MP Biomedicals, Solon, OH, USA) with either Pierce ECL Plus Western Blotting Substrate or SuperSignal West Femto reagent (Thermo Fisher Scientific, Waltham, MA, USA).
5
Immunoblotting of Protein Lysates
6
CYP1A1 and CYP1B1 Induction Assay
7
Western Blot Analysis of YY1 and Vinculin
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Total proteins were extracted with Laemmli lysis buffer (4% SDS; 20% glycerol; 10% 2-mercaptoethanol; 0.004% bromophenol blue; 0.125M Tris–HCl) and sonicated on a Bioruptor Sonication System (Diagenode, UCD-200). After 5 min denaturation at 95°C, the samples were loaded into a 4–12% gradient polyacrylamide gel (Invitrogen) for SDS-PAGE electrophoresis and transferred onto Invitrolon PVDF membranes (Invitrogen). The membranes were blocked for 1h with 5% milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) and incubated overnight at 4°C with antibodies targeting YY1 (1:500, mouse sc-7341, H-10, Santa Cruz Biotechnology) or VINCULIN (1:2000, mouse V9131, Sigma Aldrich) proteins. Proteins of interest were detected using a Peroxidase-conjugated antibody (Goat anti-mouse, 1:10 000, Sigma Aldrich) with the Pierce ECL Western blotting substrate (Thermo Scientific).
8
Histone H3 Citrullination Analysis
9
Exosome and hNSC Protein Analysis by Western Blot
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Proteins from exosomes and hNSCs were separated by SDS polyacrylamide gel electrophoresis on NuPage® 10% Bis-Tris polyacrylamide gels following manufacturer’s guidelines (Life Technologies), then transferred to Invitrolon™ PVDF membranes (Life Technologies), blocked using WesternBreeze blocker/diluent (Life Technologies) and probed with primary mouse monoclonal anti-CD63 and anti-CD81 (2 μg/ml, Life Technologies), previously described exosome enriched markers [23 (link)], and anti-c-MYC monoclonal antibody (1:100 dilution, Santa Cruz Biotechnology Inc), a hNSC marker. Proteins from untreated and scrambled miRNA (AllStars negative control siRNA AF 488, Qiagen), hsa-miR-1246 mimic (Qiagen), and exosome treated HeLa were separated and blotted as described above, and probed with primary rabbit polyclonal anti-FAM53C (1:100, Sigma) and primary mouse monoclonal α-tubulin (1:1000, Sigma). Proteins were detected using anti mouse (1:1000, Pierce Biotechnology Inc.) or anti–rabbit (1:1000, Cell Signaling Technology) horseradish peroxidase-conjugated secondary antibody accordingly and developed with enhanced chemiluminescence reagent (Thermo Scientific). Western blot images were captured using BioRad FluorS Imaging Unit and densitometry carried out using ImageJ software (National Institutes of Health).
10
Quantification of Citrullinated Histone H3
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