After treatment, hPDLSCs were washed with cold PBS and lysed in RIPA lysis buffer with the supplied with protease/phosphatase inhibitor cocktail. Protein concentrations were measured by BCA protein assay kits. Total protein (25 μg) was fractionated with SDS-PAGE gels and transferred onto PVDF membranes. The PVDF membranes were blocked with 5% skim milk and incubated with primary antibodies at 4°C overnight. Rabbit anti-Bcl2 antibodies (1: 1000), rabbit anti-Bax antibodies (1: 1000), rabbit anti-Phospho-cleaved caspase3 antibodies (1: 500), rabbit anti- caspase3 antibodies (1: 1000), rabbit anti-bmp2 antibodies (1: 1000), rabbit anti-Oct4 antibodies (1: 1000), rabbit anti-Sox2 antibodies (1: 1000), rabbit anti- Runx2 antibodies (1: 1000), rabbit anti-MyD88 antibodies (1: 1000), rabbit anti-NF-κB p65 antibodies (1: 500), rabbit anti-Phospho-NF-κB p65 antibodies (1: 1000), rabbit anti-TLR4 antibodies (1: 1000), and rabbit anti- GAPDH antibodies (1: 1000) were obtained from Cell Signaling Technology. After washing 3 times with PBS, appropriate secondary antibodies were applied for 2 h, and signals were analyzed using the Tanon-5200 Chemiluminescence Imager with enhanced chemiluminescence Western blotting substrate (Millipore, Billerica, MA).
Rabbit anti gapdh antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Rabbit anti-GAPDH antibody is a primary antibody that specifically recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a common housekeeping gene used as a loading control in western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Roti load1 buffer, by Carl Roth (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Mouse anti topoisomerase 1, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti bax antibody, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ecl prime, by GE Healthcare (2 mentions)
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Roche (2 mentions)
Odyssey clx, by LI COR (2 mentions)
Bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
Rat anti ha antibody, by Merck Group (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Enhanced chemiluminescence technique, by Thermo Fisher Scientific (2 mentions)
66 protocols using rabbit anti gapdh antibody
1
Western Blot Analysis of Stem Cell Signaling
2
Subcellular Fractionation and Protein Analysis
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Subcellular fractionations were performed per the manufacturer's instructions
(Thermo Scientific, Waltham, MA, USA). Briefly, cells at 80–90% confluence were
lysed in a series of buffers containing protease inhibitors (25X) with CERI (250
μl), CERII (11 μl), or NER (100 μl). Centrifugation steps were performed to
obtain a non-nuclear fraction and an intact nuclear pellet, followed by further
lysing to isolate the nuclear fraction. 30 μg of non-nuclear and nuclear
fractions were utilized for Western blot analysis. Mouse anti-topoisomerase I
(Santa Cruz Biotechnology Santa Cruz, CA) and rabbit anti-GAPDH antibodies (Cell
Signaling Technology, Inc., Danvers, MA) were used to ensure the integrity of
nuclear and cytoplasmic fractions, respectively. Rabbit anti-Calnexin (Santa
Cruz Biotechnology Santa Cruz, CA) was utilized as a control to ensure that the
nuclear fraction was not contaminated with endoplasmic reticulum.
(Thermo Scientific, Waltham, MA, USA). Briefly, cells at 80–90% confluence were
lysed in a series of buffers containing protease inhibitors (25X) with CERI (250
μl), CERII (11 μl), or NER (100 μl). Centrifugation steps were performed to
obtain a non-nuclear fraction and an intact nuclear pellet, followed by further
lysing to isolate the nuclear fraction. 30 μg of non-nuclear and nuclear
fractions were utilized for Western blot analysis. Mouse anti-topoisomerase I
(Santa Cruz Biotechnology Santa Cruz, CA) and rabbit anti-GAPDH antibodies (Cell
Signaling Technology, Inc., Danvers, MA) were used to ensure the integrity of
nuclear and cytoplasmic fractions, respectively. Rabbit anti-Calnexin (Santa
Cruz Biotechnology Santa Cruz, CA) was utilized as a control to ensure that the
nuclear fraction was not contaminated with endoplasmic reticulum.
3
Insulin-Mediated Cell Signaling Assay
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Bovine serum albumin (BSA), Hank's balanced salt solution (HBSS), RPMI 1640 medium, and collagenase V were obtained from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, USA). A mouse anti-insulin antibody was purchased from Abcam (Cambridge, MA). Rabbit anti-Bax, rabbit anti-Bcl-2, rabbit anti-caspase-3, and rabbit anti-GAPDH antibodies as well as secondary antibodies were obtained from Cell Signaling Technology (Beverly, USA). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased from Beyotime Institute of Biotechnology (Nantong, China).
4
Subcellular Fractionation and Validation
5
Western Blot Analysis of SIRT1 and GAPDH
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Western blotting and antibody detection were performed following the methods reported by Kawakami et al. [47 (link)]. Briefly, C2C12 cells were lysed in RIPA buffer, and the concentration of the extracted protein was measured. Proteins (20 μg) were separated using 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were first blocked using 3% BSA (for SIRT1) or 5% skim milk (for GAPDH) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h. Subsequently, they were incubated overnight at 4°C with rabbit anti-SIRT1 antibodies (Cat. No. 3931; 1:1000 in 3% BSA/TBS-T; Cell Signaling Technology, Danvers, MA, USA) or rabbit anti-GAPDH antibodies (Cat. No. 2118; 1:5000 in 5% skim milk/TBS-T; Cell Signaling Technology). After washing with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Cat. No. 7074S, Cell Signaling Technology) for SIRT1 (1:1000 in 5% skim milk) or for GAPDH (1:5000 in 5% skim milk), for 1 h at 20–25 °C. Immunoreactive bands were detected using an ECL Prime (GE Healthcare, Chalfont St. Giles, UK). Band intensities were quantified using the ImageJ, with values normalized to that of GAPDH.
6
FTL and GAPDH Protein Expression
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Western blot was performed as described previously (22 (link)). The primary reagents were used: rabbit anti-FTL (dilution, 1:1,000; Abcam, UK), rabbit anti-GAPDH antibodies (dilution, 1:1,000; Cell Signaling Technology, Inc., USA), horseradish peroxidase (HRP)–conjugated anti-rabbit IgG (dilution, 1:2,000; Cell Signaling Technology, Inc., USA), ECL Chemiluminescent HRP Substrate (Merck Millipore, USA), and BCA Protein Assay Kit (Beijing Biosynthesis Biotechnology Co., China). The gray levels of bands were analyzed by ImageJ software. The relative gray level was represented by relative IntDen (relative IntDen = the integrated band density (IntDen) of FTL/IntDen of GAPDH).
7
Glutamate-Induced Apoptosis Pathways in Astrocytes
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After astrocytes were treated with 10 mM glutamate and different doses of morphine for 24 h, the cells were lysed by RIPA lysis buffer (Beyotime, Haimen, China) according to the protocol. Western blot was carried out as previously described with minor modification [42 (link)]. The primary antibodies used in experiments were: rabbit anti-cleaved caspase-8, rabbit anti-cleaved caspase-9, rabbit anti-cleaved caspase-3, rabbit anti-phosphor-eIF2α, rabbit anti-eIF2α, rabbit anti-ATF4, mouse anti-CHOP, rabbit anti-IRE1α, rabbit anti-XBP-1, rabbit anti-phosphor-JNK and rabbit anti-GAPDH antibodies (Cell Signaling Technology, Beverly, MA, USA), mouse anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were purchased from Jackson Laboratory (Sacramento, CA, USA). Each blot was repeated at least three times, the optical density of each band was measured by Image J software (National Institutes of Health, Bethesda, MD, USA).
8
Western Blotting for Protein Detection
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Western blotting was performed as described previously (Oppermann et al. 2019 (link)). Briefly, 30 µg of protein was separated on a 4–20% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) gradient gel (Bio-Rad, Munich, Germany) and was transferred to a PVDF (polyvinylidene difluoride) membranes (Low Fluorescence Membrane Opti Blot, Abcam, Cambridge, UK). The primary antibodies used were: mouse anti-DOCK4 (Santa Cruz Biotechnology, Heiderlberg, Germany #sc-100718 1:500), rabbit anti-GAPDH antibody (Cell Signaling; #2118 1:5000) and mouse anti-ACTB (Novus Biologicals #NB600-501, 1:5000). The secondary antibodies employed (red fluorescent IRDye 680RD goat anti-mouse and green fluorescent IRDye 800CW goat anti-rabbit; both diluted 1:10,000 in TBST) were purchased from LI-COR (LI-COR Biosciences, Lincoln, USA). Membranes were scanned using an Odyssey Imaging System (LI-COR, Bad Homburg, Germany), and band intensities were determined by the Image Studio 5 software (LI-COR).
9
Adipogenic Differentiation of Cells
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Dulbecco minimum essential medium (DMEM) high glucose, fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin, were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). Adipogenic induction medium (AIM) was provided by ScienCell (Carlsbad, CA, USA). All the primers used in this study were synthesized by Invitrogen Life Technologies. Oil Red O, Hoescht 33258, Dexamethasone, β-Glycerophosphate and ascorbic acid were provided by Sigma Alrich (Sigma Alrich, St Louis, MO, USA). Mouse anti-PPARγ antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Rabbit anti-adipocyte protein2 (AP2) antibody was provided by Abcam (MA, USA), Rabbit anti-lipoprotein lipase (LPL) and rabbit anti-GAPDH antibody were obtained from Cell Signaling Technology (Beverly, MA, USA). CellTiter 96® AQueous One Solution Cell Proliferation Assay was obtained from Promega (Madison, WI, USA). BCIP/NBT ALP Color Development Kit was purchased from Beyotime (Shanghai, China). Enhanced chemiluminescence (ECL) reagent was provided by Pierce Technology (Pierce, USA).
10
Filaggrin Protein Expression Analysis
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