The expression of proteins was determined by western blot as previously described (18 (link)). The concentration of protein was detected using a BCA kit (Beyotime, Shanghai, China). Then, 30 μg of total protein was loaded onto 8% SDS polyacrylamide gels and transferred to the polyvinylidene fluoride membrane. The membrane then was blocked by 5% BSA (Sigma) for 1 h and incubated with primary antibodies at 4°C overnight. The next day, the membrane was washed with PBS for three times and then followed by incubation with HRP-labeled goat anti-rabbit IgG (Abcam, America) secondary antibody at 37°C for 1 h. Signals were detected by using the ECL detection kit (Merck Millipore) and normalized to the expression of GAPDH. The antibodies included Total insulin receptor substrate (IRS)-1 (ab52167, 1:1,000, Abcam), phosphorylated IRS-1 (p-IRS-1) at PY896 (sc-560, 1:500, Santa Cruz), total IRS-2 (ab52606, 1:1,000, Abcam), phosphorylated IRS-2 (p-IRS-2) at S731 (sc-1555-R,1:500, Santa Cruz), total PI3K (ab154598,1:1,000, Abcam), phosphorylated PI3K (p-PI3K) at P85 (ab191606, 1:1,000, Abcam), phosphorylated Akt (p-Akt) at Ser-473 (ab192623, 1:1,000, Abcam), GAPDH (ab9485, 1:1,000, Abcam). The absorbance of each band was quantitated using the Image Pro Plus Software.
Ab154598
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab154598 is a lab equipment product. It serves a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab38449, by Abcam (7 mentions)
Ab8805, by Abcam (6 mentions)
Ab182651, by Abcam (5 mentions)
Ab191606, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Ab221144, by Abcam (2 mentions)
Ab20663, by Abcam (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Ab18075, by Abcam (2 mentions)
P gsk3β ab75814, by Abcam (2 mentions)
Goat anti rabbit igg secondary antibodies ab205718, by Abcam (2 mentions)
Gene specific primers, by Sangon (2 mentions)
Ab181602, by Abcam (2 mentions)
Ecl detection kit, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Hrp labeled goat anti rabbit igg, by Abcam (1 mentions)
Ab192623, by Abcam (1 mentions)
Ab52167, by Abcam (1 mentions)
Sc 560, by Santa Cruz Biotechnology (1 mentions)
16 protocols using ab154598
1
Western Blot Analysis of IRS and PI3K Signaling
2
Protein Extraction and Western Blot Analysis
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Total cellular protein was extracted using lysis buffer, and protein levels were quantified using a BCA kit (ASPEN Biotechnology, Wuhan, China). Proteins were separated on a 10% SDS-PAGE before being probed with the appropriate primary antibodies for CD9, CD63 TSG101, PI3Kγ (#ab154598, Abcam, Cambridge, UK, 1:500), PIK3R1 (#4257, CST, MA, US, 1:1000), P-Akt (#4060, CST, MA, US, 1:1000), IL-17 (#sc-374218, Santa Cruz, Texas, US, 1:500), PTEN (#ab267787, Abcam, Cambridge, UK, 1:2000) and Bcl-2 (#ab182858, Abcam, Cambridge, UK, 1:1000) and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were then probed with secondary antibodies, and protein was identified using a LiDE110 scanner (Canon, Japan). GAPDH (#ab37168, Abcam, Cambridge, UK, 1:10,000) was used for normalization, and the AlphaEaseFC software was used to measure protein band density.
3
Protein Expression Analysis of VEGF, PI3K/Akt Pathway
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The protein contents of vascular endothelial growth factor A (VEGFA), PI3K, p-PI3K, Akt and p-Akt were evaluated by western blot and the detailed protocol as described previously (Song et al. 2019 ). In brief, 20 µg of protein isolated from cells were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and membrane transfer, followed by blocking in 5% non-fat milk. After that, the membranes were incubated with primary antibodies at 4 °C overnight, and then incubated with horseradish peroxidase (HRP)-conjugated IgG (ab205718; 1:10,000; Abcam, Cambridge, MA, USA) for 2 h at room temperature. The antibodies were as follows: anti-VEGFA (ab214424; 1:1000; Abcam), anti-cleaved caspase-3 (ab184787; 1:1000, Abcam), anti-PI3K (ab154598; 1:1000; Abcam), anti-p-PI3K (#4228; 1:600; Cell Signalling Technology, Boston, MA, USA), anti-Akt (ab8805; 1:1000; Abcam), anti-p-Akt (ab18206; 1:1000; Abcam) and anti-β-actin (ab8226; 1:1000; Abcam). Enhanced chemiluminescence reagent (ECL; Sigma) was applied to detect the protein bands. For in vitro cell samples, tests were performed with three biological replicates and three technical replicates. For animal tissue samples, tests were performed with six biological replicates and three technical replicates.
4
Western Blot Analysis of Neuronal Signaling
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Primary cortical neurons or brain tissues were lysed with RIPA buffer containing phosphatase and protease inhibitors. After measurement with BCA kits, the proteins were denatured by boiling for 10 min and then loaded on 8-12% SDS‒PAGE gels, followed by transfer from the gels to activated PVDF membranes. After blocking with 5% nonfat dry milk, the membranes were incubated with the following primary antibodies: anti-PI3K (ab154598, 1:2000), anti-p-Akt (ab38449, 1:1000), anti-Akt (ab283852, 1:2500), anti-GRP78 (ab21685, 1:3000), anti-p-PERK (ab192591, 1:1000), anti-PERK (ab79483, 1:2500), and anti-p-IRE1α (ab124945, 1:2500) were obtained from Abcam, USA and anti-IRE1α (#3294, 1:2500), anti-ATF6 (#65880, 1:2000), anti-CHOP (#5554, 1:2000), anti-Caspase12 (#35965, 1:2500), anti-Caspase3 (#9579, 1:1000), anti-Bcl-2 (#3869S, 1:2000) and anti-Bax (#5023, 1:2500) were supplied by Cell Signaling Technology, U.S.A. Anti-β-actin antibody (GB15004, 1:3000) was purchased from Servicebio Technology (China). After incubation with the primary antibodies overnight, the membranes were washed three times with TBST and incubated with secondary anti-rabbit HRP antibody (GB1213, 1:5000, Servicebio Technology, China). Enhanced chemiluminescence was used to visualize the membranes, and the analysis was performed using ImageJ software.
5
Western Blot Analysis of Protein Expression
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The protein concentration was detected by bicinchoninic acid assay. After mixing with loading buffer, the proteins were denatured by boiling. Next, the proteins were separated by electrophoresis and transferred into polyvinylidene difluoride membranes. Subsequently, the membranes were incubated with the primary antibodies and then the secondary antibody goat anti-mouse anti-IgG H&L (HRP) (1/2000, ab205719, Abcam). Then, the membranes were developed using the enhanced chemiluminescence. The levels of Ki67 (1/1000, ab16667, Abcam, Cambridge, MA, USA), Cleaved Caspase-3 (20 µg/mL, ab32042, Abcam), Bcl-2 (1/500, ab692, Abcam), RRM2 (1 µg/mL, ab57653, Abcam, Cambridge, MA, USA), AKT (1/500, ab8805, Abcam), phosphorylated AKT (p-AKT, 1/500, ab38449, Abcam), phosphatidylinositol 3-kinase (Pl3K) (1/500, ab154598, Abcam), p-Pl3K (1/500, ab182651, Abcam) were determined, with GAPDH (1/500, ab8245, Abcam) acting as the internal reference. The quantification of band intensity was processed using Image-Pro Plus 6.0 software (Olympus, Tokyo, Japan).
6
Melanocytic Pathway Regulation Protocol
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Polymyxin B was obtained from USP Reference Standards (lot no. R046V0). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, and 0.25% trypsin were purchased from GIBCO Invitrogen (CA, USA). All chemicals and reagents were purchased from MedChemExpress (NJ, USA) at analytical grade. Gene-specific primers were obtained from Sangon Biotech (Shanghai, China). MITF (ab20663), TYR (ab18075), DCT (ab221144), PI3K (ab154598), p-PI3K (ab182651), GSK3β (ab32391), p-GSK3β (ab75814), β-catenin (ab16051), p-β-catenin (ab75777), p-CREB (ab32096), CREB (ab32515) and β-actin (ab8227) primary antibodies and goat anti-rabbit IgG secondary antibodies (ab205718) were purchased from Abcam (Cambridge, MA, USA).
7
Western Blot Analysis of PI3K/AKT Pathway
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RIPA lysate was added to the cells to obtain total protein, and protein concentration was calculated by a BCA protein quantitative kit (Beyotime, Shanghai, China). Appropriate protein dosage was electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto PVDF membranes. Then, the membranes were blocked with 5% skim milk at 25°C for 2 h, and the primary antibody diluent of the corresponding protein was incubated at 4°C overnight. Thereafter, the corresponding secondary antibody was incubated at room temperature for 2 h. Afterwards the bands were incubated with ECL luminescence solution (Thermo Fisher Scientific, USA) and placed in a chemiluminescence imaging system to expose for visualization. The relative expression of each protein was deconstructed using ImageJ software (National Institutes of Health). Antibody information is as follows: PI3K (ab154598, 1 : 2000, Abcam, UK), p-PI3K (ab278545, 1 : 1000), AKT (ab8805, 1 : 500, Abcam, UK), p-AKT (ab38449, 1 : 1000, Abcam, UK), GAPDH (ab181602, 1 : 10,000, Abcam, UK).
8
Immunoblotting Protocol for Protein Analysis
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Immunoblotting was conducted following the procedures by the published study [28 (link)]. Briefly, cells were collected by centrifugation, and total protein was extracted with RIPA buffer. Proteins were separated by 5%-12% SDS-PAGE gels (80–120 V), transferred to the PVDF membrane at 180 V, and blocked in 5% skimmed milk in TBST overnight. The membranes were incubated with the following primary antibodies (mouse or rabbit anti-human) for 4 h at room temperature: anti-human AKAP3 (1:500; Invitrogen, PA5-109,376), anti-PTEN (1:500; Beyotime, AP686), anti-PI3K (1:500; Abcam, ab154598), anti-AKT (1:500; Abcam, EPR16798), anti-mTOR (1:500), anti-pAKT (phospho T308) (1:200; Abcam, ab38449), anti-pmTOR (phospho S2448) (1:200; Abcam, ab131538), and anti-β-actin (1:1000; Beyotime, AF0003) antibodies. After three washes with TBST, membranes were incubated with Horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG secondary antibody (1:5000) for 2 h at room temperature. Protein bands were visualized using the ECL luminescent solution, photographed, and quantitated using the Image J software.
9
Western Blot Analysis of Angiogenic Factors
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The expression levels of PI3K, AKT, eNOS, and VEGF were detected by Western blot. Cells were grinded in liquid nitrogen and washed with precooled PBS buffer. Then, the samples were split in cold Ripa cracking buffer (Beyotime, Shanghai, China). The total protein content was measured by the BCA kit (Aapplygen, Beijing, China). Then the total protein was separated by 10% gel SDS-PAGE (120 V electrophoresis 90 min). The protein was transferred to the PVDF membrane (Bio-Rad, California, USA) (50 V, 120 min). It was sealed in a closed solution containing 5% skim milk. Then, PDVF membrane was combined with anti-PI3K (ab154598, Abcam, Cambridge, USA), anti-AKT (ab8805, Abcam, Cambridge, USA), anti-eNOS (ab76198, Abcam, Cambridge, USA), and anti-VEGF (Abcam, Cambridge, Ma, USA) (1:1000 dilution) were incubated overnight at 4 °C and then, incubated at room temperature for 1 h with 1:5000 diluted HRP coupling second antibody. The protein bands were visualized by the ECL kit. All antibodies were purchased from Abcam (USA). GAPDH was used as the internal parameter, and the gray level of protein bands was detected by imagePD software.
10
Protein Expression Analysis of PI3K/AKT/mTOR Pathway
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The treated cells were fully lysed to extract proteins and prepare suspensions, 10% SDS-PAGE were prepared, electrophoresed at 40V for 4-5h, then transferred to
nitrocellulose membranes and blocked, antibody incubation was performed, and finally visualized and image analysis was carried out. PI3K/p-PI3K (Abcam, ab154598, UK), AKT/p-AKT (Abcam,
ab38449, UK), mTOR/p-mTOR (Abcam, ab134903, UK), Bcl-2 (Invitrogen, PA5-11379, USA), Bax (Invitrogen,
14-6997-82, USA), GAPDH (Invitrogen, 39-8600, USA).
nitrocellulose membranes and blocked, antibody incubation was performed, and finally visualized and image analysis was carried out. PI3K/p-PI3K (Abcam, ab154598, UK), AKT/p-AKT (Abcam,
ab38449, UK), mTOR/p-mTOR (Abcam, ab134903, UK), Bcl-2 (Invitrogen, PA5-11379, USA), Bax (Invitrogen,
14-6997-82, USA), GAPDH (Invitrogen, 39-8600, USA).
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