The antibodies used in this study were as follows: EGFR (1:50; # ab52894), HER3 (1:25; # ab5470), HER4 (1:150; # ab19391), VEGFR3 (1:100; # ab27278), VEGF-C (1:50; # ab135506), Wnt5a (1:100; # ab72583), Beta-Catenin (1:100; # ab32572), p-Akt1 (1:100; # ab32505), Akt1 (1:50; #ab59380) were purchased from Abcam company, UK. HER2 (1:100, #4290) was purchased from Cell Signaling Technology, Inc., USA. EphrinA1 (1:100; # sc-911) and EphA3 (1:100; # sc-920) were purchased from Santa Cruz Biotechnology, USA. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako EnVision, USA).
P akt1
Manufactured by Abcam
Sourced in United Kingdom, United States
P-AKT1 is a recombinant protein that corresponds to a phosphorylated form of the AKT1 protein. AKT1 is a serine/threonine-protein kinase that plays a key role in the regulation of cell survival, growth, proliferation, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
β catenin, by Abcam (3 mentions)
P mtor, by Abcam (2 mentions)
P gsk 3β, by Abcam (2 mentions)
Twist, by Abcam (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Gsk 3β, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Vegfr3, by Abcam (1 mentions)
Ephrina1, by Santa Cruz Biotechnology (1 mentions)
Vegf c, by Abcam (1 mentions)
Epha3, by Santa Cruz Biotechnology (1 mentions)
Wnt5a, by Abcam (1 mentions)
Enhanced chemiluminescence method, by Thermo Fisher Scientific (1 mentions)
P mapk, by Abcam (1 mentions)
P pten, by Abcam (1 mentions)
P pi3k, by Abcam (1 mentions)
Protease inhibitor, by Santa Cruz Biotechnology (1 mentions)
12 protocols using p akt1
1
Comprehensive Antibody Panel for Signaling
2
Protein Expression Analysis in Synovium
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The total synovial proteins were extracted using the protein extraction kit (Beibo Corp., Shanghai, China) and then resolved by electrophoresis. Next, the proteins were transferred from the gel onto the PVDF membrane (Millipore Corp., USA) using the semidry method (Tianneng Corp., China). The PVDF membranes were incubated with respective MAPK, PI3K, Akt1,PTEN,p-MAPK, p-PI3K, p-Akt1, and p-PTEN antibodies (dilution 1 : 1000; Abcam Biotechnology, Cambridge, UK) for 2 h at RT, followed by incubation with the following secondary antibodies for 1 h at RT: antimouse secondary antibody (dilution 1 : 5000. Zhongshan Goldenbridge Biotechnology Corp., Beijing, China) for MAPK, PI3K, Akt1, PTEN, p-MAPK, p-PI3K, p-Akt1, and p-PTEN. The enhanced chemiluminescence method (Thermo) was used to develop the bands, and the images were obtained.
3
Western Blot Analysis of AKT Pathway
4
BMSC Protein Extraction and Western Blot
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According to a previously established method [31 (link)], RIPA (Beyotime, Shanghai, China) and PMSF (Beyotime, Shanghai, China) were prepared in working solution that was used to extract the total protein of BMSCs. Proteins were electrophoresed and resolved by SDS-PAGE gels, and PVDF membranes (Millipore, Billerica, MA, USA) were used to transfer the proteins on the gels. Primary antibodies against RUNX2 (Wanleibio, Shenyang, China), OPN (Wanleibio, Shenyang, China), AKT1 (Abcam, Cambridge, UK), P-AKT1 (Abcam, Cambridge, UK), and GAPDH (ABclonal, Wuhan, China) were diluted 1:1000 and incubated with PVDF membranes overnight at 4°C. After washing thrice with TBST, the PVDF membranes were incubated with the corresponding secondary antibodies (ABclonal, Wuhan, China) at a dilution ratio of 1:5000 for 1 h. Finally, proteins were visualized by chemiluminescence, and quantitatively analyzed using ImageJ software.
5
Protein Extraction, Quantification, and Western Blotting
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Total protein extraction and quantification utilized radioimmunoprecipitation assay (RIPA) buffer (Sigma Aldrich, USA) and a BCA Protein Assay Kit (Thermo, USA) respectively. SDS–PAGE (Thermo, USA) separated 20 μg of protein lysates, subsequently transferred to polyvinylidene fluoride membranes (Millipore, Germany). Specific antibodies against GAPDH (1:2000, ZENBIO, China), DNAJC12 (1:2000, Abcam, UK), β-Actin (1:2000, Cloud-Clone Corp, China), HSP70 (1:2000, Abcam, UK), SLC7A11 (1:2000, Abcam, UK), GPX4 (1:2000, Abcam, UK), caspase 3 (1:2000, Proteintech, US), p-AKT1 (1:2000, Abcam, UK), AKT1 (1:2000, Proteintech, US), and DDDDK tag (1:2000, Abcam, UK) were employed to incubate with the cut bands.
6
Investigating Wnt, EGF, and TGF-β Signaling
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For in vitro studies, stock solutions of MLN8237 (20 mmol/L), FH535 (10 mmol/L) and LY294002 (20 mmol/L) were prepared in DMSO. Stock solutions of recombinant human Wnt3a (100 μg/ml), EGF (100 μg/ml) and TGF-β1(100 μg/ml) were diluted in phosphate-buffered saline (PBS).
Dimethyl sulfoxide (DMSO), LY294002 and EGF were purchased from Sigma. FH535 was purchased from Merck. Wnt3a was purchased from abnova. MLN8237 was purchased from selleck.
AURKA, β-catenin, AKT1, p-AKT1, GSK-3β, p- GSK-3β, Twist, H3K4 me3/AC and H3K27 me2/me3/AC antibodies were purchased from Abcam. E-cadherin, N-cadherin, H3K4 me1/me2 and H3K27 me1 antibodies were purchased from Cell Signaling Technology. The Ki67 antibody was purchased from Santa Cruz Biotechnology. The H3 antibody was purchased from Ray Antibody. The GAPDH antibody was purchased from Zhongshan Bio Corp.
Dimethyl sulfoxide (DMSO), LY294002 and EGF were purchased from Sigma. FH535 was purchased from Merck. Wnt3a was purchased from abnova. MLN8237 was purchased from selleck.
AURKA, β-catenin, AKT1, p-AKT1, GSK-3β, p- GSK-3β, Twist, H3K4 me3/AC and H3K27 me2/me3/AC antibodies were purchased from Abcam. E-cadherin, N-cadherin, H3K4 me1/me2 and H3K27 me1 antibodies were purchased from Cell Signaling Technology. The Ki67 antibody was purchased from Santa Cruz Biotechnology. The H3 antibody was purchased from Ray Antibody. The GAPDH antibody was purchased from Zhongshan Bio Corp.
7
Western Blot Analysis of EMT Markers
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UBE2C, AURKA, β-catenin, AKT1, p-AKT1, GSK-3β, p-GSK-3β, Slug, Snail and Twist antibodies were purchased from Abcam (Cambridge, UK). E-cadherin, N-cadherin and p-AURKA antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The GAPDH antibody was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Western blot analysis reagents were purchased from Sigma, PVDF membranes were purchased from Millipore Corp. (Bedford, MA, USA) and RIPA lysis buffer was purchased from Beijing Taike Biotechnology. Lentiviruses containing an UBE2C and AURKA inhibitor sequences (Lenti-si-UBE2C and Lenti-si-AURKA) or negative control (Lenti-NC) were obtained from Shanghai GeneChem Co., Ltd. (Shanghai, China).
8
Astragaloside IV for Inflammatory Bowel Disease
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Astragaloside IV was purchased from Dalian Meilun Co., Ltd. (Shanghai, China, purity > 98%). 5-amino salicylic acid (5-ASA) was purchased from MCE Ltd. (New Jersey, USA, purity ≥ 98%). DSS was purchased from MP Biomedicals (San Diego, USA). Sodium carboxymethyl cellulose (CMC-Na) and FITC-Dextran were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were from Shanghai Enzyme Union Co., Ltd. (Shanghai, China). β-actin antibody was purchased from Proteintech Co., Ltd. (Wuhan, China). Villin, zonula occludens-1 (ZO-1), Occludin, epithelial cell adhesion molecule (EpCAM), AKT1, p-AKT1, PI3K, and p-PI3K antibodies were from Abcam (Cambridge, UK) and CST Corporation (Boston, USA). Goat anti-rabbit IgG H&L (HRP) was obtained from Servicebio Co., Ltd. (Wuhan, China). Alexa Fluor 488-AffiniPure Goat Anti-Human IgG + IgM (H + L) was from Jackson Immuno Research (Pennsylvania, USA). LY294002 and YS49 were purchased from MCE Ltd. (New Jersey, USA).
9
Heme Oxygenase 1 Regulation in HUVECs
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HUVECs were harvested using trypsin, lysed with RIPA buffer, and the supernatants were collected by centrifugation. The protein content in the RIPA lysates is quantified, and the concentration was determined using the BCA Protein Assay reagent kit. Proteins were fractionated using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes; the membranes were blocked for 1 h in 5% skim milk before being incubated with antibodies against heme oxygenase 1 (HMOX1), p-AKT1, and AKT1 (Abcam, UK) at 4 °C overnight. The membranes were then washed three times in TBST (Tris-buffered saline, 0.1% Tween 20) for 10 min each. Then, each membrane was incubated with anti-rabbit IgG H&L for 1 h at room temperature. Finally, each membrane was washed again with TBST and bound protein was visualized using an enhanced chemiluminescence (ECL) Western blotting detection kit (GE Healthcare, Chicago, IL, USA); signals were observed using GeneGnome (Syngene, Bangalore, India). For HMOX1 gene silencing and overexpression, the cells were transfected with plasmids designed by OBiO Technology (Shanghai, China).
10
Protein Expression Analysis by Western Blot
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Western blotting was performed using the standard protocol published elsewhere. The following primary antibodies were used: β-catenin (Sigma, MA1-301, 1:2000), p-β-catenin (Cell Signaling Technology, 9561, 1:500), β-actin (Cell Signaling Technology, 3700S, 1:2000), ERK1/2 (Cell Signaling Technology, 9107, 1:2000), p-ERK1/2 (Cell Signaling Technology, 4370, 1:2000), periostin (Invitrogen, PA5-34641, 1:500), p- GSK-3β (Cell Signaling Technology, 5558, 1:2000), GSK-3β (Cell Signaling Technology, 12456, 1:2000), BRAF (Cell Signaling Technology, 9433, 1:500), CRAF (Cell Signaling Technology, 9422, 1:500), Akt1 (Abcam, ab32505, 1:1000), p-Akt1 (Abcam, ab66138, 1:1000), and Lamin B (Invitrogen, 50-554-010, 1:1000). Membranes were incubated with primary antibodies overnight at 4°C, washed three times using TBST (Trisbuffered saline, 0.1% Tween 20), and then incubated with IRDye 800CW donkey anti-rabbit IgG secondary antibody (LI-COR, 926-32213, 1:2000) or IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, 926-68072, 1:2000). The membranes were then washed again using TBST and processed for scanning and visualization using an Odyssey CLx imaging system (LI-COR, model #9140). Protein bands were quantified using ImageJ as published before 75 .
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