Immunoblotting was performed to detect the protein expression of hBMSCs. hBMSCs were treated with different concentrations of Gd-BG dissolution for different time points. Briefly, the cells were washed with PBS and then collected in cell lysis buffer. Protein concentration was determined using the bicinchoninic acid assay kit (Cell Signaling Technology, Danvers, MA, USA). For this, 10 µg of the extract was electrophoresed in 10% sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were treated with the relative antibodies. Primary antibodies, including anti-β-catenin, anti-p-Akt, anti-total-Akt, anti-p-GSK3β, and anti-total-GSK3β, were provided by Cell Signaling Technology. The membranes were then incubated with a secondary antibody (Cell Signaling Technology). After chemiluminescence, LEICA DM 4000 was used to detect the target bands. The results were normalized to the loading control (GAPDH; BioTNT, Shanghai, China). All samples were measured in triplicate.
Anti total gsk 3β
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Anti-total GSK-3β is a primary antibody used to detect the expression of the total glycogen synthase kinase-3 beta (GSK-3β) protein. GSK-3β is a serine/threonine protein kinase that plays a crucial role in diverse cellular processes. This antibody can be used in various immunological techniques, such as Western blotting, to analyze the levels of total GSK-3β in biological samples.
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Lab products found in correlation
Anti β catenin, by Cell Signaling Technology (3 mentions)
Anti p gsk 3β, by Cell Signaling Technology (3 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti total akt, by Cell Signaling Technology (2 mentions)
Rabbit anti caspase 1, by Santa Cruz Biotechnology (2 mentions)
Mouse anti ser473akt, by Cell Signaling Technology (2 mentions)
Rabbit anti ser9 gsk 3β, by Abcam (2 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Rabbit anti total akt, by Cell Signaling Technology (2 mentions)
Rabbit anti nlrp3, by Abcam (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti phospho gsk3β ser9, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Bicinchoninic acid assay kit, by Cell Signaling Technology (1 mentions)
Mouse anti tubulin, by Abcam (1 mentions)
Anti phospho ser 21 9 gsk3α β, by Cell Signaling Technology (1 mentions)
Anti phospho ser thr pka substrate, by Cell Signaling Technology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
10 protocols using anti total gsk 3β
1
Immunoblotting of Gd-BG Effects on hBMSCs
2
Immunoblot Analysis of Signaling Proteins
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Equal amounts of total protein extracts were separated by SDS-PAGE and electrotransferred to nitrocellulose membrane (GE-Healthcare Europe, Milan, Italy). Membranes were probed with rabbit anti-NLRP3 (Abcam, Cambridge, UK), rabbit anti-caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Ser473Akt (Cell Signaling Technology, Danver, MA, USA), rabbit anti-total Akt (Cell Signaling Technology, Danver, MA, USA), rabbit anti-Ser9 GSK-3β (Abcam, Cambridge, UK), anti-total GSK-3β (Cell Signaling Technology, Danver, MA, USA), anti-Ser660 PKC and total PKC (Santa Cruz Biotechnology, Dallas, TX, USA), SOD2 (Novus Biologicals, Centennial, CO, USA), and NRF2 (Thermo Fisher Scientific, Whaltam, MA, USA) followed by incubation with appropriate HRP-conjugated secondary antibodies (BioRad). Proteins were detected with Clarity Western ECL substrate (BioRad, California, USA) and quantified by densitometry using analytic software (Quantity-One, BIO-RAD Image Lab Software.6.0.1.). Results were normalized with respect to densitometric value of mouse anti-tubulin (Abcam, Cambridge, UK), and autoradiograms showing statistically significant differences in terms of gel-loading homogeneity were excluded from the following biomarkers analyses.
3
Bisphenol A metabolic effects
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Bisphenol A (BPA), 4,4′-Sulfonyldiphenol (BPS), dihydrorhodamine 123 (DHR) and merocyanine M540 were from Sigma-Aldrich (St Louis, MO, USA); propidium iodide (PI), SYBR-14, 5,5′,6,6′–tetrachloro-1,1′,3,3′ tetraethylbenzymidazolyl carbocyanine (JC-1), Yo-Pro-1 and CellROX Green probe were from Thermo Fisher Scientific (Eugene, OR, USA); DC Protein Assays and 2X Laemmli Sample Buffer were from Bio-Rad (Hercules, CA, USA); and Intercept (TBS) blocking buffer and IRDye 800RD and 680RD secondary antibodies were from LI-COR Biotechnology (Bonsai Lab, Alcobendas, Spain). Furthermore, the anti-phospho (Ser/Thr) PKA Substrate (#9624), anti-phospho (Ser 21/9) GSK3α/β (#9331) and anti-total GSK3β (#9332) polyclonal antibodies were from Cell Signaling Technology, Inc. (Beverly, MA, USA); the anti-phospho-tyrosine monoclonal antibody (4G10, 05-321) was from Millipore (Burlington, MA, USA); the anti-α-tubulin monoclonal antibody (TU-02, #SC-8035) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All reagents used to prepare incubation media were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
Western Blot Analysis of Signaling Proteins
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Western blot analysis was performed according to standard methods, as previously described 10 (link), using anti-KPNA2 (Abcam, Cambridge, UK), anti-total Akt, anti-p-Akt, anti-total GSK-3β, anti-p-GSK-3β, anti-snail, anti-twist, anti-vimentin, anti-E-cadherin, anti-Fibronectin, and anti-α-catenin antibodies (Cell Signaling Technology, Danvers, MA, USA). Polyvinylidene fluoride membranes were stripped and re-blotted with an anti-β-actin monoclonal antibody (Sigma, St. Louis, MO, USA) as a loading control.
5
Inhibition of Inflammatory Pathways
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p38-MAPK inhibitor (SB203580), ERK1/2 inhibitor (U0126), JNK inhibitor (SP600125), NF-κB inhibitor (Bay 11–7082), Akt inhibitor (LY-294002), and DAPI were purchased from Sigma-Aldrich. GM-CSF was purchased from R&D Systems. Kits for Cell Proliferation Assay (MTS), DeadEnd Fluorometric TUNEL System, and TEV protease were purchased from Promega. In Situ Cell Death Detection kit and cOmplete His-Tag Purification Resin were purchased from Roche. BrdU ELISA kit was purchased from Abcam. Protein A Agarose and Alexa Fluor 488 Phalloidin were purchased from Thermo Fisher Scientific. Antibodies for Western blotting used in this study, including anti–Phospho-NF-κB p65 (Ser536), anti–total-NF-κB p65, anti–Phospho-Akt (Ser473), anti–total-Akt, anti-Phospho-GSK3β (Ser9), anti–total-GSK3β, anti–β-catenin, and anti–β-actin, were purchased from Cell Signaling Technology; anti–human TREM2 antibody for Western blotting was purchased from R&D Systems (AF1828). Antibodies for immunofluorescence including anti-Iba1 and anti–human TREM2 were purchased from Wako and Santa Cruz Biotechnology (sc-373828), respectively.
6
Western Blot Analysis of Inflammasome Signaling
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Total proteins extracts were separated by SDS-PAGE and blotted to nitrocellulose membrane (GE-Healthcare Europe, Milano, Italy). Membranes were incubated with rabbit anti-NLRP3 (Abcam, Cambridge, UK), rabbit anti-caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-GSDMDC1 (Santa Cruz Biotechnology), rabbit anti-IL-1β (Santa Cruz Biotechnology), rabbit anti-caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Tyr204 ERK1/2 (Cell Signaling Technology), rabbit anti-total ERK1/2 (Cell Signaling Technology), mouse anti-Ser473 Akt (Cell Signaling Technology), rabbit anti-total Akt (Cell Signaling Technology), rabbit anti-Ser9 GSK-3β (Abcam, Cambridge, UK), anti-total GSK-3β (Cell Signaling Technology), rabbit anti-mitochondrial transcription factor A (mtTFA) (Novus Biologicals, Cambridge, UK), mouse anti-nuclear respiratory factor-1 (NRF-1) (Santa Cruz Biotechnology), and mouse anti-sarcomeric mitochondrial creatine kinase (sMtCK) (Santa Cruz Biotechnology) and then probed with proper HRP-conjugated secondary antibodies (BioRad). Clarity Western ECL substrate (BioRad) was used for protein detection and quantification was performed by densitometric analysis (Quantity-One, Bio-Rad software). Data were normalized according to the related antitubulin densitometric values.
7
Protein Extraction and Western Blot Analysis
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Cells were lysed in lysis buffer containing 150 mM NaCl, 1.0% nonidet-P 40 (NP40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0, and a protease inhibitor cocktail (Roche Applied Science, Vienna, Austria, pH 7.4). Electrophoresis was performed as described previously7 (link).
The following primary antibodies were used for Western blot analysis: anti-CTMP, anti-vimentin, anti-Slug, anti-β-actin (1:1,000; Cell Signaling Technology Inc.), anti-E-cadherin, anti-Snail (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-total Akt, -phopho-Akt (Ser 473), anti-total GSK3β, and -phospho-GSK3β (Ser 9) (Cell Signaling Technology). Following incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies, immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection.
The following primary antibodies were used for Western blot analysis: anti-CTMP, anti-vimentin, anti-Slug, anti-β-actin (1:1,000; Cell Signaling Technology Inc.), anti-E-cadherin, anti-Snail (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-total Akt, -phopho-Akt (Ser 473), anti-total GSK3β, and -phospho-GSK3β (Ser 9) (Cell Signaling Technology). Following incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies, immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection.
8
Western Blot Analysis of hBMSC Proteins
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The proteins were obtained from the hBMSCs using a cell lysis buffer supplemented with a proteinase inhibitor. The amount of protein was measured using the BCA protein assay kit (Cell Signaling Technology, Danvers, MA). A total of 20 μg of protein were resolved on SDS-PAGE gels and then transferred to PVDF membranes. The protein was blocked with 5% milk in Tris-buffered saline 0.1% Tween (TBST). The membranes were incubated with primary antibodies. Primary antibodies including anti-β-catenin, anti-Runx2, anti-p-GSK3β, and anti-total-GSK3β were provided by Cell Signaling Technology, Shanghai, China. The membranes were then incubated with an anti-rabbit and anti-mouse secondary antibody (Cell Signaling Technology). After chemiluminescence, the LEICA DM 4000 was used to detect the target bands. The protein levels were normalized by β-actin (Cell Signaling Technology).
9
Protein Analysis by Western Blotting
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For protein analysis, cells were lysed in RIPA buffer. The protein concentration was quantified with a BCA Protein Quantitation Assay Kit (Beyotime Biotech., Shanghai, China). Equal amounts of protein were loaded and separated by 10% SDS‐PAGE and then transferred onto a PVDF membrane (Millipore Corp., Billerica, MA, USA). The membranes were incubated overnight with appropriate primary antibodies at 4°C. Bound antibodies were then visualized using horseradish peroxidase‐conjugated secondary antibodies. A quantitative analysis was performed by using ImageJ software (US National Institutes of Health). For the antibody information, the antibody against PPARα was purchased from Proteintech (Chicago, IL, USA). Anti‐phospho‐JNK (Thr183/Tyr185), anti‐total JNK, anti‐phospho‐ERK1/2 (Thr202/Tyr204), anti‐total ERK1/2, anti‐total p38, anti‐phospho‐GSK3β (Ser9) and anti‐total GSK3β antibodies were obtained from Cell Signalling Technology (Danvers, MA, USA). The antibody against anti‐phospho‐p38 (Thr180/Tyr182) was purchased from Bioworld Technology, Inc (Nanjing, China). The antibody against GAPDH was derived from Kangcheng Biotech (Shanghai, China).
10
Western Blot Analysis of LV Proteins
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