The protein samples were prepared from cell lysates and resolved by SDS‐PAGE. Subsequently, protein samples were transferred to Immobilon polyvinyldifluoride membranes and blocked with 4% bovine serum albumin for 1 hour at room temperature. The blots were probed with rabbit anti‐human antibodies against candidate signal pathway proteins—pAKT (1:1000; GeneTex), AKT (1:1000; GeneTex), MMP2 (1:1000; GeneTex), and MMP9 (1:1000; Abcam)—for 2 hours at room temperature. After three washes with tris‐buffered saline containing Tween 20, the blots were subsequently incubated with a donkey anti‐rabbit peroxidase‐conjugated secondary antibody (1:3000; Cell Signaling Technology) for 1 hour at room temperature. Protein levels on the blots were detected with enhanced chemiluminescence using Kodak X‐OMAT LS film (Eastman Kodak) and quantified using a computing densitometer and ImageQuant (Molecular Dynamics).
P akt
Manufactured by GeneTex
Sourced in United States
P-AKT is a laboratory equipment product manufactured by GeneTex. It is a phospho-specific antibody that detects the phosphorylation of the AKT protein at the Ser473 residue. The core function of P-AKT is to enable the analysis and quantification of phosphorylated AKT in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
P erk, by GeneTex (3 mentions)
P fak, by GeneTex (3 mentions)
Gapdh, by GeneTex (2 mentions)
Dasatinib, by Bio-Techne (2 mentions)
Mouse monoclonal antibodies of egfr, by Santa Cruz Biotechnology (2 mentions)
Rabbit polyclonal antibody of c src, by Santa Cruz Biotechnology (2 mentions)
Recombinant human cxcl1, by Thermo Fisher Scientific (2 mentions)
Vcam 1, by GeneTex (2 mentions)
P iκbα, by GeneTex (2 mentions)
Cxcr2, by GeneTex (2 mentions)
P p85α, by GeneTex (2 mentions)
P ikkα β, by GeneTex (2 mentions)
Ikkα β, by GeneTex (2 mentions)
β actin, by GeneTex (2 mentions)
Imagequant, by GE Healthcare (1 mentions)
Mmp 9, by Abcam (1 mentions)
X omat ls film, by Kodak (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
11 protocols using p akt
1
Quantitative Western Blot Analysis
2
Protein Extraction and Western Blot Analysis
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For protein extraction, 1 × 106 cells were lysed in 200 μL RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS) containing Complete Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN, USA). Cell lysates were centrifuged at 14,000× g for 30 min at 4 °C, then supernatant was harvested. Proteins were quantified by use of the Bio-Rad DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA), separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blotted with antibody for UBE2C (H00011065-M01, Abnova), mTOR (#2983, Cellsignal, Danvers, MA, USA.), p-mTOR (Cellsignal, #2971), AKT (Genetex, GTX121937), p-AKT (GTX128414, Genetex, Ivine, CA, USA), PI3K (Genetex, GTX112994), p-PI3K (Cellsignal, #4228), GAPDH (Genetex, GTX627408) and anti-β-actin (Thermofisher, #MA5-15739).
3
Establishment of GC Xenograft Model
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Male 4-5 weeks-old BALB/c nude mice (Shanghai SLAC Laboratory Animal Center, Shanghai, China) were used to establish GC xenograft model. BGC823 cells (2×106/100μl) were implanted by subcutaneous injected into the right axillary fossa of mouse. After one week, the animals were randomly divided into four groups (six mice per group) and given: (1) control (sterile physiological saline, i.p., every other day), (2) OMT (30mg/kg, i.p., every other day), (3) CDDP (5mg/kg, i.p., every other day), (4) OMT + CDDP (30mg/kg + 5mg/kg, i.p., every other day). After 24 days, mice were sacrificed and xenografts tumors were harvested and weighed. The tumor volume and inhibition ratio were tested as described before 19 (link).
The paraffin-embedded sections (5μm thick) were prepared and stained with hematoxylin and eosin (H&E) or immunohistochemically with p-AKT, p-ERK or Ki-67 (mouse anti-Ki67 antibody; GeneTex Inc, Irvine, California) according to previously reported protocols 19 (link), 20 (link).
The paraffin-embedded sections (5μm thick) were prepared and stained with hematoxylin and eosin (H&E) or immunohistochemically with p-AKT, p-ERK or Ki-67 (mouse anti-Ki67 antibody; GeneTex Inc, Irvine, California) according to previously reported protocols 19 (link), 20 (link).
4
Antibody and Kinase Inhibitor Sources
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Rabbit polyclonal antibodies of Hic-5, p-Src, p-AKT, and AKT were purchased from GeneTex (Irvine, CA, USA) whereas mouse monoclonal antibodies of EGFR, c-Met, Her2, Her3, p-JNK, JNK and rabbit polyclonal antibody of c-Src were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Src kinase inhibitors PP1 were from Merck (Darmstadt, Germany) and dasatinib was from Tocris Bioscience (Bristol, UK).
5
Antibody and Inhibitor Reagents for Signaling
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Rabbit polyclonal antibodies of Hic-5, phosphorylated Src (p-Src), and AKT (p-AKT) and Ki67 were purchased from GeneTex (Irvine, CA, USA), whereas mouse monoclonal antibodies of EGFR, c-Met, Her2, Her3, phosphorylated JNK (p-JNK) and rabbit polyclonal antibody of c-Src were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The Src kinase inhibitor dasatinib was from Tocris Bioscience (Bristol, UK). The glycolysis inhibitor 3-Bromopyruvate (3-BrP) was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
6
Assessing Vascular Remodeling in Lung Disease
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The extent of muscularization of small arteries was assessed following immunochemistry staining for α‐smooth muscle actin (α‐SMA) (Abcam, Cambridge, UK). Meanwhile, smooth muscle cells of small lung arteries exhibiting proliferation were assessed by immunostaining with proliferating cell nuclear antigen (PCNA) (ProteinTech, Wuhan, China) and osteopontin (OPN) (ProteinTech, Wuhan, China). Furthermore, the expression of KLF4 and P‐AKT was assessed by immunostaining with antibodies against KLF4 (ProteinTech, Wuhan, China) and P‐AKT (Gene Tex, San Antonio, USA).
7
Western Blot Quantification of Key Signaling Proteins
8
Protein Co-Immunoprecipitation Assay
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Most chemicals used and Protein A-agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibody for CCT-β was purchased from Abcam (Cambridge, MA, USA) for the co-IP assay. For western blots, the specific antibodies for GAPDH (100118), CCT-α (16399), CCT-β (112283), CCT-γ (33073), CCT-δ (33536), CCT-ε (110167), CCT-ζ (105148), CCT-η (101347), CCT-θ (115466), XIAP (111202), β-catenin (101435), P-β-catenin (S675) (132611), P-β-catenin (Ser33/Ser37/Thr41) (132605), Slug (121924), Snail (100754), Twist (127310), E-cadherin (100443), MUC1 (100459), N-cadherin (112734), MMP2 (104577), MMP9 (100458), AKT (121973), P-AKT (128414), GSK-3β (111192), P-GSK-3β (50090), Survivin (100052), NF-kB p65 (102090), fibronectin (112794), β1 (112971), Src (134412), P-Src (54701), FAK (50489), and P-FAK (129840) were from GeneTex (Irvine, CA, USA). The catalog numbers are in parentheses.
9
Molecular Mechanisms of CXCL1/CXCR2 Signaling
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All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The CXCL1, CXCR2, VCAM-1, p-FAK, FAK, p-p85α, p85α, p-Akt, Akt, p-IKKα/β, IKKα/β, p-IκBα, IκBα, p-p65, p65, and β-Actin specific anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase antibodies were purchased from GeneTex International Corporation (Hsinchu City, Taiwan). Recombinant human CXCL1 was purchased from PeproTech (Rocky Hill, NJ, USA). Short hairpin RNA (shRNA) plasmid for CXCR2 and VCAM-1 were obtained from the National RNAi Core Facility Platform (Taipei, Taiwan). All siRNAs were ON-TARGETplus siRNAs, obtained from Dharmacon Research (Lafayette, CO, USA). The NF-κB luciferase report plasmid, pSV-β-galactosidase vector, and luciferase assay kit were purchased from Promega (Madison, WI, USA).
10
Signaling Pathway Regulation Assay
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Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for CXCL1, CXCR2, VCAM-1, p-FAK, FAK, p85α, p-p85α, Akt, p-Akt, p-IKKα/β, IKKα/β, p-IκBα, IκBα, p-p65, p65, and β-Actin were purchased from GeneTex International Corporation (Hsinchu City, Taiwan). Recombinant human CXCL1 was purchased from PeproTech (Rocky Hill, NJ, USA). Short hairpin RNA (shRNA) plasmid for knocking down gene expression was purchased from the National RNAi Core Facility Platform (Taipei, Taiwan). All siRNAs were ON-TARGETplus siRNAs, purchased from Dharmacon Research (Lafayette, CO, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
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