Protein expression was determined by western blot. All experiments were performed at 72 hours to ensure adequate protein expression. Cells were washed twice with cold PBS and then cell lysates were collected on ice with 10μg phosphatase inhibitor cocktail (Roche). Equal amount of protein lysates from each condition were separated by using NuPAGE 15 well 4–12% Bis-Tris protein gel (Thermo Fisher, MA), then transferred onto nitrocellulose membrane. The membrane was cut into three and probed separately with E-cad antibody (Invitrogen, 334000), p53 antibody (cell signaling, 2527S) and GAPDH antibody (GeneTex, 627408), followed by secondary antibody (Abcam, 205718 & 97040). Here we used GAPDH as our loading control, because the variation of GAPDH expression among different donors/transfections was less than 1 fold (data not shown here). These antibodies have been confirmed by manufacture and previous studies [11 (link),43 ].
Gapdh antibody
Manufactured by GeneTex
Sourced in United States
The GAPDH antibody is a laboratory reagent used to detect the presence and measure the levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in various biological samples. GAPDH is a widely expressed and highly conserved enzyme involved in the glycolytic pathway. The GAPDH antibody is a specific and sensitive tool for researchers studying GAPDH expression and its role in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Roche (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Amphotericin b, by Thermo Fisher Scientific (3 mentions)
Mini protease inhibitor cocktail, by Roche (2 mentions)
Phosstop, by Merck Group (2 mentions)
Mab5232, by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin g, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Amersham ecl select, by GE Healthcare (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Las 1000plus, by Fujifilm (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
Caspase 1 antibody, by Merck Group (1 mentions)
Gtx213110 01, by GeneTex (1 mentions)
19 protocols using gapdh antibody
1
Protein Expression Analysis by Western Blot
2
Quantifying SLC37A2 Protein Levels in AoSMCs
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Total protein was collected from AoSMC samples cultured under the same conditions used for real-time PCR analysis. These samples were lysed using Tris-buffered saline (pH 7.5) supplemented with 5% NP-40 and a 1% protease inhibitor cocktail. The protein concentration samples was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), and the concentration was adjusted. For western blotting, 10 µg of protein was separated in a 10% polyacrylamide gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was treated with SLC37A2 antibody (Novus Biologicals, Centennial, CO) or GAPDH antibody (GeneTex, Irvine, CA) as the primary antibodies, incubated with Rabbit IgG Horseradish peroxidase-conjugated antibody (R&D Systems, Minneapolis, MN) as the secondary antibody, and visualized using Amersham ECL Select (GE Healthcare, Buckinghamshire, England) and luminescent image analyzer (LAS-1000 plus, FUJIFILM, Tokyo, Japan). The band was quantified using ImageJ.
3
Immunoblotting Analysis of Signaling Proteins
4
Western Blot Assay for Protein Analysis
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Western blot assay was performed as described previously39 with the specific antibodies listed below: Anti‐Asialoglycoprotein Receptor 1 antibody (ASGR1‐Ab, ab254262, Abcam Inc., Cambridge, MA), Phospho‐Stat3 (Tyr705)(D3A7) XP Rabbit mab (pSTAT3 Ab, #9145, Cell Signaling Inc., Danvers, MA), Anti‐h/m/rSTAT3 purified mouse monoclonal IgG (STAT3 Ab, #MAB1799, R&D Systems Inc., Minneapolis, MN), GAPDH antibody (GTX100118, GeneTex Inc., CA), Mouse IgG antibody (HRP) (GTX213111‐01, GeneTex Inc., CA), Rabbit IgG antibody (HRP) (GTX213110‐01, GeneTex Inc., CA).
5
Western Blot Analysis of BRM and Phospho-Rb
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Following the treatment with flavonoids or the transfection experiments, cells were harvested and total protein was extracted using a urea-based lysis buffer as described previously [17 (link), 25 ]. A rabbit polyclonal anti-BRM antibody was used for the detection of BRM at a dilution of 1:500 [17 (link)]. Mouse anti-phospho Rb (BD Biosciences, San Jose, California) was used at 1:250 for the detection of phsopho-Rb protein. Appropriate secondary antibodies (GE Healthcare, UK) were used at a dilution of 1:2000. GAPDH antibody (GeneTex Inc., Irvine, CA, USA) was used as the loading control.
6
Western Blot Analysis of TRAIL Protein
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After cells were infected, the culture supernatants were centrifuged and concentrated. The cells were then lysed. Proteins were separated and then transferred onto polyvinylidenedifluoride (PVDF) membranes (Millipore, USA). After blocking, the membranes were incubated with TRAIL antibody (Abcam, UK), GAPDH antibody (GeneTex, USA), or GFP antibody (GeneTex, USA) overnight at 4°C. After the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Bio-Rad, USA), the bands were detected using Western ECL Blotting Substrate (Bio-Rad, USA).
7
Quantifying CHIKV Structural and Nonstructural Proteins
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As described earlier,61 (link) BHK-21 cells infected
with CHIKV were harvested
after 24 h of drug treatment as per the procedure mentioned inSection 4.8 . Harvested
cells were lysed with RIPA buffer as well as with the help of an ultrasonicator.
After quantification, protein-containing cell lysate samples were
separated on 10% SDS-polyacrylamide gel by loading 50 μg of
protein in each well and run for 3 h at 100 V. Separated proteins
were subsequently transferred onto a polyvinylidene difluoride (PVDF)
membrane. To determine the CHIKV structural and nonstructural proteins,
the PVDF membrane was blocked with 5% skim milk and washed and then
the membrane was incubated with E2 (The Native Antigen Company) and
nsP2 (Antibody Research Corporation) primary antibodies at a dilution
factor of 1:2500. The membrane was then washed three times with washing
buffer, and GAPDH (GAPDH antibody was obtained from GeneTex) was used
as a loading control. The PVDF membrane was then subsequently incubated
for 2 h with secondary antibody (goat anti-mouse HRP-conjugated) at
a dilution of 1:5000. The membrane was then washed and blots were
developed with developing reagents. The band intensities of the CHIKV
structural and nonstructural proteins were measured from three independent
experiments.
with CHIKV were harvested
after 24 h of drug treatment as per the procedure mentioned in
cells were lysed with RIPA buffer as well as with the help of an ultrasonicator.
After quantification, protein-containing cell lysate samples were
separated on 10% SDS-polyacrylamide gel by loading 50 μg of
protein in each well and run for 3 h at 100 V. Separated proteins
were subsequently transferred onto a polyvinylidene difluoride (PVDF)
membrane. To determine the CHIKV structural and nonstructural proteins,
the PVDF membrane was blocked with 5% skim milk and washed and then
the membrane was incubated with E2 (The Native Antigen Company) and
nsP2 (Antibody Research Corporation) primary antibodies at a dilution
factor of 1:2500. The membrane was then washed three times with washing
buffer, and GAPDH (GAPDH antibody was obtained from GeneTex) was used
as a loading control. The PVDF membrane was then subsequently incubated
for 2 h with secondary antibody (goat anti-mouse HRP-conjugated) at
a dilution of 1:5000. The membrane was then washed and blots were
developed with developing reagents. The band intensities of the CHIKV
structural and nonstructural proteins were measured from three independent
experiments.
8
Phosphorylation of Presenilin 1 in Microglia
9
Immunoblotting for Inflammatory Markers
10
Protein Extraction and Western Blot Analysis
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The gills excised from one individual were pooled as one sample for total protein extraction. Protein extraction and Western blot were performed following a previous protocol [62 (link)]. A total of 20 μg of total protein was loaded for Western blot. Custom rabbit Ncc2 polyclonal antibody against the N-terminal domain (IKKSRPSLDVLRNPPDD) of zebrafish Ncc2 (400 ng/mL) [22 (link)], the GAPDH antibody (GeneTex, Hsinchu City, Taiwan) (1:5000 dilution), and Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Invitrogen, Waltham, MA, USA) (1:5000 dilution) were used for immunoblotting. WesternBright ECL (Advansta, San Jose, CA, USA) was used to produce signals. All images were obtained by a UVP ChemStudio PLUS Imaging System (Analytik Jena, Jena, Germany) and quantitated by Fiji [61 (link)].
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