Total proteins used for Western blotting analysis were extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) supplemented with protease inhibitors (Roche, Basel, Switzerland). With the help of the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA), the total protein was quantified. The Western blotting system was established using a Bio-Rad Bis-Tris Gel system (Shanghai, P.R. China) according to the manufacturer’s instructions. GAPDH antibody was purchased from Sigma-Aldrich, which was used as the loading control. Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1,000. Primary antibody was incubated with the membrane at 4°C overnight, followed by washing and incubation with secondary antibody tagged with horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride (PVDF) membrane carrying the blotted proteins and antibodies was transferred into the Bio-Rad ChemiDoc™ XRS System (Shanghai, P.R. China), and then 200 μl of Immobilon Western Chemiluminescent HRP Substrate (Millipore) was added to cover the membrane surface. The protein signals were captured using Image Lab™ Software (Bio-Rad, Shanghai, P.R. China).
Gapdh antibody
Manufactured by Merck Group
Sourced in United States, Germany, China
The GAPDH antibody is a laboratory reagent used for the detection and quantification of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a commonly used reference or housekeeping protein in various biochemical and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Bca protein assay, by Thermo Fisher Scientific (5 mentions)
β actin antibody, by Merck Group (4 mentions)
Protein assay kit, by Bio-Rad (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Odyssey imaging system, by LI COR (3 mentions)
Nupage, by Thermo Fisher Scientific (3 mentions)
Socs3, by Cell Signaling Technology (3 mentions)
Goat anti rabbit dylight 800, by Thermo Fisher Scientific (3 mentions)
Goat anti mouse dylight 680, by Thermo Fisher Scientific (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Roche (3 mentions)
Protease inhibitors cocktail, by Roche (3 mentions)
Lds sample buffer, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
144 protocols using gapdh antibody
1
Western Blot Protein Quantification
2
Characterization of DNA Damage Response Proteins
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Human cell lines U2OS, HEK293T and HCT116 were purchased from ATCC. MDCI-, H2AX-, RNF8-, 53BP1- deficient MEFs and wild-type MEF were gereruously supplied by Dr. Xiaochun Yu described previously[19 (link)]. Human cell lines and MEF cells were maintained in DMEM medium with 10% fetal calf serum and cultivated at 37°C in 5% CO2 (v/v). Cells were lysed with NETN buffer (contains 0.5% NP40,50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM EDTA) with Roche Protease Inhibitor Cocktail. Immunoprecipitation and western blotting were performed following standard protocol as described previously. Rabbit anti-RNF138 antibody is purchased from Santa Cruse, anti-glutathione s-transferase (GST) antibody, monoclonal anti-β-actin antibodies and GAPDH antibody were purchased from Sigma. Mouse monoclonal anti-GFP antibody and Phospho-(Ser/Thr) ATM/ATR Substrate antibody were purchased from Cell Signaling Technology. RPA antibody was purchased from Abcam. γH2AX, RAD51, RNF8, MDC1, BRCA1, Rabbit anti-53BP1 antibody was kind gifts from Dr. Xiaochun Yu.
3
Protein Extraction and Immunoblotting
4
Quantification of Transcription Factor Activation
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot analysis were performed using standard protocols as described in [77 (link)]. The protein content in whole-cell extracts from K4IM cells was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA). For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium). The GAPDH antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the horseradish peroxidase conjugated secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Proteins were visualized by chemiluminescence (Supersignal West chemiluminescent substrate; Pierce) using a Bio-Rad ChemiDoc apparatus [74 (link)].
5
Examining Involucrin Expression in HaCaT Cells
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In order to examine the expression of involucrin in the HaCaT cell line, cells were washed with PBS, then collected and solubilized in RIPA buffer on ice for 30 min. The lysate was centrifuged at 12,000 rpm for 15 min at 4 °C to remove cellular debris and the supernatant protein concentration was determined by the BCA protein assay (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The proteins were then added to 5 × loading buffer and incubated at 95 °C for 5 min before being subjected to standard western blotting.
The primary antibody against involucrin (Abcam, Cambridge, UK) were diluted at 1:500, ATP5B antibody (Abcam, Cambridge, UK) was diluted at 1:400, β-actin antibody (Sigma, Germany) was diluted at 1:10,000, and GAPDH antibody (Sigma, Germany) was diluted at 1:5000. Immunolabeled proteins were then visualized using a goat anti-mouse antibody linked to horseradish peroxidase (1:10,000, Sigma, Germany) and an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA). Protein levels in each lane were normalized to that of β-actin or GAPDH.
The primary antibody against involucrin (Abcam, Cambridge, UK) were diluted at 1:500, ATP5B antibody (Abcam, Cambridge, UK) was diluted at 1:400, β-actin antibody (Sigma, Germany) was diluted at 1:10,000, and GAPDH antibody (Sigma, Germany) was diluted at 1:5000. Immunolabeled proteins were then visualized using a goat anti-mouse antibody linked to horseradish peroxidase (1:10,000, Sigma, Germany) and an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA). Protein levels in each lane were normalized to that of β-actin or GAPDH.
6
Characterization of Anti-TRIM44 Antibody
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Anti‐DYKDDDDK tag antibody (anti‐flag antibody) was purchased from Wako Pure Chemical Industries (Osaka, Japan) and anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody was purchased from Sigma‐Aldrich Japan (Tokyo, Japan). Anti‐TRIM44 polyclonal antibody was manufactured at our laboratory as described elsewhere in the previous report.20 This antibody is an affinity purified rabbit polyclonal antibody raised by immunizing rabbits with a glutathione S‐transferase (GST) fusion protein with amino acids of full‐length mouse TRIM44 protein as an antigen. The antiserum was absorbed with GST‐bound resin and anti‐GST antibody was removed. The non‐absorbed components were further purified by using an affinity column filled with the antigen. The quality and characterization of these antibodies were confirmed by Western blotting analysis of the human TRIM44‐transfected 293 T cells.
7
Western Blot Analysis of TRPV1 in Spinal Cord
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The spinal cord was lysed in ice-cold RIPA buffer with a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO). Tissues were homogenized using a tissue homogenizer. Spinal cord samples were loaded into the 10% polyacrylamide gels and then were electrotransferred onto a nitrocellulose membrane. The membranes were blocked with 10% non-fat dry milk in PBS with 0.3% Tween for 1 hour and then incubated overnight at 4°C with rabbit anti-TRPV1 antibody (1:500; Alomone Labs, Jerusalem, Israel), or GAPDH antibody (1:5000; Sigma Aldrich, St. Louis, MO) diluted in PBS. The membrane was then incubated in horseradish peroxidase-conjugated anti-rabbit IgG (1:5000; Santa Cruz Biotechnology, Dallas, TX) for 2 hours at room temperature. The antigen–antibody complexes were visualized using an enhanced chemiluminescence detection reagent (Bio-Rad Laboratories, Hercules, CA). Bands were scanned using a densitometer (GS-700; Bio-Rad Laboratories, Hercules, CA), and quantification was performed using NIH Image J software.
8
Regulation of hOCTN1 expression in RASF
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To down-regulate hOCTN1 expression, RASF were transfected with either 40 nM hOCTN1 siRNA (Sigma-Aldrich; GACAAUUUACUGUGAGUUA) or non targeting scramble siRNA (Invitrogen; nonsilencing control siRNA) using a N-TER™ Nanoparticle siRNA Transfection System® (Sigma) according to the manufacturer’s instructions. The transfection efficiency was verified by PCR. The dependence of saracatinib biological impact on hOCTN1 expression in RASF was assessed at the molecular level by determination of c-Abl activity. RASF transfected with hOCTN1- or scramble-siRNA were incubated with 1 μM or 10 μM saracatinib and 10 ng/ml PDGF for 10 min, and total extracts were examined for c-Abl activation by Western blotting using anti-phospho Abl (Cell signaling, Danvers, USA; diluted 1:500) and GAPDH antibody (Sigma-Aldrich). The part of membrane containing GAPDH, as determined from molecular weight markers, was cut and the expression of GAPDH was detected as loading control. Western blots were quantified by ImageJ software (NIH, Bethesda, MD, USA).
9
Immunoprecipitation and Western Blot Analysis of Succinylated GAPDH in Ganoderma lucidum
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The mycelia of G. lucidum G.260125–1 were disrupted and the soluble proteins were prepared [6 (link)]. The extracted proteins (1 mg) were incubated with or without 1 μg of GAPDH antibody (Sigma-Aldrich) at 4 °C for 6 h. The mixture was then supplemented with 20 μl protein A agarose beads (GE Healthcare) followed by incubating at 4 °C for 12 h. After separated from the mixture by a centrifugation at 4 °C and 6,000×g for 1 min, the agarose beads were washed for 3 times, and the boiled SDS-PAGE sample buffer was used to elute the binding proteins on the agarose beads [7 (link)]. The eluted proteins were isolated on 12% gel using SDS-PAGE and then electrotransferred onto a polyvinylidene difluoride membrane followed by detection using GAPDH antibody (1:10,000 dilution) and succinyllysine antibody (1:2000 dilution, PTM Biolabs) [6 (link), 7 (link)], respectively. Finally, the Western blot signal was detected using an immunoblotting detection kit (ThermoFisher).
10
Mitochondrial Dynamics in Cell Signaling
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Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS) and trypsin were purchased from Thermo Fisher. Notch1, Mfn1, Mfn2, OPA1, Drp1, Fis1, Cytochrome C and COX IV antibodies were purchased from Cell Signaling Technology. GAPDH antibody was purchased from Sigma. ATP detection kit was from Beyotime Biotechnology. Enhanced chemiluminescence kit was purchased from Thermo Fisher Scientific.
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