The protein was extracted using lysis buffer, and protein concentration was measured via BCA assay kits. After electrophoresis and membrane transfer, the bands were incubated with the primary antibody including PAK1 (1/1000, ab223849, Abcam) and GAPDH (1/2500, ab9485, Abcam) at 4°C overnight, then cultured with secondary antibody at room temperature for 2 h. The chemiluminescence system (Bio-Rad, USA) imaging system was performed to measure all bands.
Ab223849
Manufactured by Abcam
Sourced in United States
Ab223849 is a primary antibody product from Abcam. It is a recombinant monoclonal antibody targeted against a specific protein. The antibody is supplied in a liquid form and is suitable for various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab76293, by Abcam (2 mentions)
Dasatinib, by Selleck Chemicals (2 mentions)
Ipa 3, by Bio-Techne (2 mentions)
Frax597, by Bio-Techne (2 mentions)
β actin, by Merck Group (2 mentions)
Chemiluminescence system, by Bio-Rad (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab31624, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
Ab184993, by Abcam (1 mentions)
Ab240678, by Abcam (1 mentions)
Ab178945, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ab32536, by Abcam (1 mentions)
Bicinchoninic acid kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab133462, by Abcam (1 mentions)
6 protocols using ab223849
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Protein Targets
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Total protein extracts from tissues and cells were prepared using RIPA lysis buffer (Beyotime, Haimen, China) supplemented with PMSF (Beyotime). Sample protein concentration was measured using the bicinchoninic acid kit (Beyotime). Proteins were separated on 8–12% polyacrylamide gels by electrophoresis and subsequently transferred onto polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% nonfat milk for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. After washing with Tris-buffered saline/0.1% Tween 20 (TBST), the membranes were incubated with secondary for 2 hours at room temperature and immunoreactive bands then detected using an enhanced chemiluminescence reagent. Antibodies against TGS101 (ab125011), CD206 (ab64693), iNOS (ab178945) α-SMA (ab240678), PAK1 (ab223849), collagen I (ab34710), collagen III (ab184993), p-p65 (ab31624), p65 (ab32536), p-IKKβ (ab114243), and p-IκBα (ab133462) were obtained from Abcam (Cambridge, MA, USA).
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from the cerebral cortex or cultured cells using the Membrane and Cytosol Protein Extraction Kit (Beyotime, China) and measured with the BCA Protein Assay Kit (Beyotime, China). Fifty micrograms protein per sample was separated through 10% ExpressPlus™ Gels (GenScript, the USA) and then transferred to Polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in buffer, the membranes were incubated overnight at 4°C with the following antibodies: Caspase‐1 (89332, CST), Cleaved Caspase‐3 (ab214430, Abcam), COX‐2 (12282, CST), p‐JNK (ab124956, Abcam), JNK (ab179461, Abcam), p‐P38 (4511, CST), P38 (8690, CST), BAX (ab32504, Abcam), Bcl‐2 (ab182858, Abcam), MMP‐2 (87809S, CST), MMP‐9 (ab228402, Abcam), Catalase (ab76110, Abcam), Cu/Zn SOD (ab13498, Abcam), β‐actin (4970, CST), CD63 (EXOAB‐CD63A‐1, SBI), CD81 (EXOAB‐CD81A‐1, SBI), CD9 (EXOAB‐CD9A‐1, SBI), Rac1 (8631, CST), Cdc42 (8747, CST), PAK‐1(ab223849, Abcam) and p‐PAK1 (ab75599, Abcam). The membranes were washed and incubated with the secondary antibodies for 2 hours, and the positive bands were detected with a chromogenic developing agent.
4
Establishment of Xenograft Model for A549 Cells
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All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Gansu Provincial Tumor Hospital. For the establishing of the xenograft model, A549 cells (1 × 107) with sh‐NC or sh‐hsa_circ_0004396 were subcutaneously injected into the nude mice (4 weeks old, 5 mice/group). At the same time, mice were checked daily with normal activities such as eating, drinking, eliminating, and ambulating. Tumor size was measured every 5 days, followed by calculation following the formula that length × width2/2. After injection for 30 days, the mice were sacrificed and the tumor weight was measured. Immunohistochemical staining assay was carried out as described previously,28 and tissue sections from the obtained tumors were incubated with antibodies specific for Ki67 (0.5 μg/ml, ab15580; Abcam) and (1:100, ab223849; Abcam).
5
PAK Kinase Inhibitor Protocols
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IPA-3 (#3622) and PIR3.5 (#4212) were purchased from Tocris Bioscience and dissolved in sterile dimethylsulfoxide (DMSO) to make 50 mM stock solutions. Working solutions were prepared by 10-fold dilution of the stock solution in 50 mM Tris, pH 8.0, immediately before use. FRAX597 (#6029) was purchased from Tocris Biosciences and dissolved in sterile DMSO to make a 10 mM stock solution. Working solution was prepared by 10-fold dilution in cell culture medium. Dasatinib was obtained from Selleckchem (S1021), and a 200 µM stock solution was made in sterile DMSO. The following antibodies were used: PAK1 (Abcam, ab223849), PAK2 (Abcam, ab76293), PAK1 (phosphoS144)+PAK2 (phosphoS141)+PAK3 (phosphoS139) (Abcam, ab40795), PAK1/2/3 (Cell Signaling, #2604), and β-actin (Sigma-Aldrich, A5441).
6
PAK Kinase Inhibitor Protocols
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IPA-3 (#3622) and PIR3.5 (#4212) were purchased from Tocris Bioscience and dissolved in sterile dimethylsulfoxide (DMSO) to make 50 mM stock solutions. Working solutions were prepared by 10-fold dilution of the stock solution in 50 mM Tris, pH 8.0, immediately before use. FRAX597 (#6029) was purchased from Tocris Biosciences and dissolved in sterile DMSO to make a 10 mM stock solution. Working solution was prepared by 10-fold dilution in cell culture medium. Dasatinib was obtained from Selleckchem (S1021), and a 200 µM stock solution was made in sterile DMSO. The following antibodies were used: PAK1 (Abcam, ab223849), PAK2 (Abcam, ab76293), PAK1 (phosphoS144)+PAK2 (phosphoS141)+PAK3 (phosphoS139) (Abcam, ab40795), PAK1/2/3 (Cell Signaling, #2604), and β-actin (Sigma-Aldrich, A5441).
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