Dexamethasone (Dex) were purchased from Selleck (Selleck Chemicals, United States). The FKBP51 shRNA plasmid (h) (sc-35380-sh), scramble shRNA (sc-108060) and transfection reagent (sc-108061) as well as the primary antibodies against FKBP51 (sc-271547) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology, Inc. (Shanghai, China). FKBP51 lentiviral expression vectors were constructed and packaged by Shanghai GeneChem BioTECH (Shanghai, China). Puromycin, Ara-C and AKT inhibitor (A6730) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Primary antibodies against phospho-GSK3β (S9) (ab75814), GSK3β (ab32391), P21 (ab109520), P27 (ab32034), p-FOXO1A (S256) (ab31339), FOXO1A (ab52857), BAX (ab32503) and BCL-2 (ab32124) were purchased from Abcam (Shanghai, China). The primary antibodies against phospho-AKT (Ser473) (4060S) and AKT (pan)(4685S) were purchased from Cell Signalling Technology, Inc. (Danvers, MA, USA). The goat anti-rabbit horseradish peroxidase-labelled secondary antibody (Z2301) was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China).
Ab75814
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab75814 is a primary antibody that recognizes the Histone H3 (tri methyl K27) protein. It is designed for use in applications such as Western Blotting and Immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab32391, by Abcam (20 mentions)
Ab32572, by Abcam (10 mentions)
Ab81283, by Abcam (7 mentions)
Ab32072, by Abcam (6 mentions)
Ab32503, by Abcam (5 mentions)
Ab32124, by Abcam (5 mentions)
Ab8805, by Abcam (5 mentions)
Ab8245, by Abcam (5 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor, by Roche (4 mentions)
Lysis buffer, by Beyotime (4 mentions)
Ab271977, by Abcam (4 mentions)
Ab51037, by Abcam (4 mentions)
Ab109094, by Abcam (4 mentions)
Ab179463, by Abcam (4 mentions)
Ab109520, by Abcam (3 mentions)
Ab134175, by Abcam (3 mentions)
Ab205718, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab38449, by Abcam (3 mentions)
32 protocols using ab75814
1
Dexamethasone Regulation of FKBP51
2
Protein Immunoblotting Assay Protocol
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Protein was extracted, separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes, followed by incubation with the appropriate concentration of various antibodies. The antibodies were as follows: anti-CARM1 (ab128851, NOVUS, NB200-342); anti-GSK3β (#9315; CST, Danvers, MA, USA), anti-GSK3β (ab75814; Abcam), anti-β-catenin (#9562; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-cyclinD1 (ab134175; Abcam), anti-PRAS40 (ab214100; Abcam), anti-ERK1/2(ab214362; Abcam), anti-Lamin B1 (#12586; CST), anti-rabbit IgG (Sigma-Aldrich), or anti-mouse IgG (Sigma-Aldrich).
3
Western Blot Analysis of EMT Markers
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Cells were collected with scrapers on ice-bath. The total protein was extracted with cell lysis buffer and protein concentration of each group was determined by bicinchoninic acid method (BCA, Beyotime, China). Forty µg proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking with skim milk, the membranes were incubated with polyclonal rabbit antibodies at 4 °C for a night and goat anti-rabbit IgG-HRP (1:5,000; E-AB-1003; Elabscience, China) at 37 °C for 1 hour. After washing the membranes with TBST for three times, the membranes were exposed and developed with Super ECL plus hypersensitive luminescence solution (Applygen, China). The relative expression of proteins was analyzed by Image J. The primary antibodies used are as follows: RGN (1:1,000; 17947-1-AP; Proteintech, USA), β-catenin (1:1,000; 51067-2-AP; Proteintech, USA), p-GSK-3β (1:1,000; ab75814; Abcam, UK), GSK-3β (1:1,000; 22104-1-AP; Proteintech, USA), MMP-3 (1:1,000; 17873-1-AP; Proteintech, USA), MMP-7 (1:800; 10374-2-AP; Proteintech, USA), MMP-9 (1:800; 10375-2-AP; Proteintech, USA), E-cadherin (1:1,000; 20874-1-AP; Proteintech, USA), N-cadherin (1:1,000; 22018-1-AP; Proteintech, USA), Vimentin (1:1,000; 10366-1-AP; Proteintech, USA), GAPDH (1:1,000; 10494-1-AP; Proteintech, USA).
4
Wnt2b-Mediated Regulation of Amyloid-β Signaling
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Synthetic human Aβ1‐42 peptide was purchased from GL Biochem , Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco , mouse recombinant Wnt2b (rWnt2b) was purchased from Cusabio, 1,1,1,3,3,3‐Hexafluoro‐2‐propanol (HFIP) was purchased from Sigma‐Aldrich . The primary antibodies used were rabbit anti‐Wnt2b (ab178418; Abcam), mouse anti‐β‐catenin (ab32572; Abcam); rabbit anti‐phospho‐β‐catenin (Thr41/Ser45) (9565 S; Cell Signaling Technology); rabbit anti‐GSK3β (ab32391; Abcam); rabbit anti‐phospho‐GSK3β (Ser9) (ab75814; Abcam); rabbit anti‐BDNF (ab108319; Abcam); rabbit anti‐SDHB (A10821; ABclonal); mouse anti‐β‐actin (ab6276; Abcam); The secondary antibodies used were Goat Anti‐Rabbit IgG (HRP) (ab205718; Abcam); Goat Anti‐Mouse IgG (HRP) (ab6789; Abcam); Donkey anti‐Rabbit IgG (ab150073; Abcam).
5
Protein Expression Analysis in LUAD Cells
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Total protein was extracted from LUAD cells using RIPA buffer (Beyotime, Beijing, China) containing protease inhibitor and phosphatase inhibitor. The protein concentrations were detected by BCA assay (Beyotime, Beijing, China). An equal amount of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, CA, USA). After blocking with 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies specific for UBE2T (1:1000, ab140611, Abcam), FBLN5 (1:500, ab66339, Abcam), Bcl-2 (1:2000, ab182858, Abcam), Bax (1:1000, ab32503, Abcam), cleaved-caspase-3 (1:500, ab32042, Abcam), pro-caspase-3 (1:1000, ab32499, Abcam), p-ERK (1:2000, Cell Signaling Technology, #4370), ERK (1:1000, Cell Signaling Technology, #4695), p-GSK3β (1:2000, ab75814, Abcam), GSK3β (1:2000, ab32391, Abcam), β-catenin (1:1000, Cell Signaling Technology, #8480), β-actin (1:1000, ab8227, Abcam) at 4°C overnight, followed by HRP-conjugated secondary antibody (1:2000, ab205718, ab6789, Abcam) for 1 h. The positive bands were detected by using an Enhanced Chemiluminescence Kit (Thermo Fisher Scientific) [32 (link)].
6
Comprehensive Protein Analysis Protocol
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Total cellular proteins were extracted using the cell lysis buffer for Western blot. The protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Bio-Rad, USA). The membranes were blocked in 5% skim milk and then incubated with a specific primary antibody and a secondary antibody, and they were then detected by enhanced chemiluminescence (ECL). The immunoblots were visualized using the Image Quant LAS 4000 digital imaging system (GE, USA). The following primary antibodies were used: Antibodies for PHLPP2 (PA5-25995) and Vimentin (PA5-2723) were obtained from Thermo Fisher. Antibodies for GSK-3β (ab131356), p-GSK-3β (ab75814), P27 (ab62364), P21 (ab109520), CyclinD1 (ab134175), E-cadherin (ab152102) and Snail (ab82846) were purchased from Abcam. Antibodies for AKT (D260001) and p-AKT (D155022) were purchased from Sangon Biotech . While the β-actin antibody and the secondary antibodies were purchased from Beyotime.
7
Antler Cell Protein Analysis
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Antler cells (10) were collected and lysed for 20 min using lysis buffer (RIPA lysis and 50 × Cocktail) (Servicebio, Wuhan, China). Protein concentration was examined using the BCA protein assay (Servicebio, Wuhan, China). The total protein (20 μg) was separated by SDS-polyacrylamide gel electrophoresis. Subsequently, proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% skimmed milk or bovine serum albumin (BSA) powder with 0.1% Tween-20 in TBS for 2 h and incubated with primary antibody at 4 °C overnight. The following antibodies were used: anti-YAP1 (GTX129151, GeneTex, San Antonin, TX, USA, 1:1000), anti-p-YAP1 (S127) (ab76252, Abcam, Cambridge, UK, 1:2000), anti-GSK3 beta (phosphor S9) (ab75814, Abcam, 1:1000), anti-PRDX2 (ab109367, Abcam, 1:2000), anti-β-catenin (ab32572, Abcam, 1:2000), anti-Wnt5a (ab179824, Abcam, 1:2000), anti-Jak2 (3230, Cell Signaling Technology (CST) Biological reagents Company Ltd., Shanghai, China, 1:1000), anti-p-JAK2 (Try1007/1008) (3771, CST, 1:1000), anti-p-STAT3 (Tyr705) (9145, CST, 1:1000) and anti-Col2a (15943, Proteintech, Wuhan, China, 1:1000). After washing, membranes were incubated with secondary antibodies for 2 h. Lastly, the membranes were incubated with ECL chemiluminescence reagent and exposed to X-ray film for the observation of protein bands.
8
Protein Extraction and Western Blot Analysis
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The extraction of total protein from cells and tissue samples was performed by radio-imunoprecipitation assay lysis buffer with 1% phenylmethanesulfonyl fluoride, 1% phosphatase inhibitor, and 1% protease inhibitor (Beyotime Biotech, China). The experimental procedure was carried out as usual. The information of antibodies is listed as follows: anti-TET3 antibody (dilution, 1:1,000; ab153724; Abcam), anti-β-catenin antibody (dilution, 1:2,000; ab32572; Abcam), anti-p-AKT (T308) antibody (dilution, 1:1,000; ab38449; Abcam), anti-p-AKT (S473) antibody (dilution, 1:1,000; ab81283; Abcam), anti-p-GSK3β (Tyr216 + Tyr279) antibody (dilution, 1:2,000; ab68476; Abcam), anti-p-GSK3β (Ser9) antibody (dilution, 1:2,000; ab75814; Abcam), anti-GSK3β antibody (dilution, 1:5,000; ab32391; Abcam), anti-AKT antibody (dilution, 1:1,000; ab8805; Abcam), and anti-GAPDH antibody (dilution, 1:5,000; ab9482; Abcam).
9
Immunofluorescence analysis of signaling proteins
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After they were treated with vehicle or dioscin, cell samples were fixed using 4% paraformaldehyde solution for 15 min at room temperature and then extracted with buffer containing 0.5% Triton X-100 for 5 min. The cells were then incubated with primary antibodies against SOX2 (1:100, #ab92494, Abcam), β-catenin (1:100, # ab22656, Abcam), phospho-Akt (Ser473) (1:100, #ab81283, Abcam), phospho-GSK3β (Ser9) (1:100, #ab75814, Abcam) at 4 °C overnight. Next, samples were incubated with secondary antibody at room temperature for 1 h. Finally, nuclei were counterstained with DAPI. Immunofluorescence was detected using laser scanning confocal microscope (Olympus FV1000).
10
Protein Expression Analysis in Ischemic Mouse Brains
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Total protein of the cortex of the ischemia side in mice brains or cultured cells was extracted using a RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Approximately equal amounts of protein were separated by 12% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% skim milk, the membranes were incubated with primary antibodies, including FGF19 (1:500, ab225942, Abcam), GSK-3β (1:500, ab93926, Abcam), phosphorylated-GSK-3β (1:500, ab75814, Abcam), Nrf2 (1:500, ab62352, Abcam), Lamin B2 (1:1000, ab8983, Abcam) and GAPDH (1:2000, ab8425, Abcam), overnight at 4 °C. Afterwards, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, ab205718, Abcam) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescent (ECL) kit.
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