The specific antibody of EGFR, p-EGFR (Tyr1068), p-PI3K, Akt, mTOR, p-mTOR (Ser2448), Met, p-Met (Y1234/1235), ERK1/2, p-ERK1/2, β-actin and β-tubulin were purchased from Cell Signaling Technology (Beverly, MA). PI3K and p-Akt (Ser473) were obtained from ABcam (Cambridge, UK). The enhanced chemiluminescence (ECL) system was from Millipore (Millipore, MA, USA). Epidermal growth factor (EGF) was purchased from Biosource International Inc. (Camarillo, CA) and dissolved in phosphate-buffered saline (PBS).
P akt ser473
Manufactured by Abcam
Sourced in United Kingdom, United States
P-Akt (Ser473) is a phosphorylation-specific antibody that recognizes Akt protein phosphorylated at serine 473. Akt is a serine/threonine protein kinase that plays a key role in multiple cellular processes, including cell proliferation, survival, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
β tubulin, by Cell Signaling Technology (2 mentions)
P pi3k, by Cell Signaling Technology (2 mentions)
P akt thr308, by Abcam (2 mentions)
P enos ser1177, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
α tubulin, by Merck Group (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Mnsod, by Merck Group (2 mentions)
Foxo3, by Cell Signaling Technology (2 mentions)
P yap, by Cell Signaling Technology (2 mentions)
Catalase, by Abcam (2 mentions)
Foxo1, by Abcam (2 mentions)
Ab54073, by Abcam (2 mentions)
Lats2, by Abcam (2 mentions)
P foxo1, by Cell Signaling Technology (2 mentions)
P enos, by Cell Signaling Technology (2 mentions)
P mst1, by Cell Signaling Technology (2 mentions)
16 protocols using p akt ser473
1
Signaling Pathway Protein Analysis
2
Western Blot Analysis of Liver Proteins
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Total protein of rat liver tissue in each group was extracted using radioimmunoprecipitation assay lysis buffer. The protein concentration was determined using a bicinchoninic acid protein concentration quantification kit (Beyotime Biotechnology, Shanghai, China). Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk in tris-buffered saline containing 0.05% Tween 20 or 1–3% bovine serum albumin and probed with primary antibodies overnight at 4°C. The primary antibodies used included IRS-1, Akt (1 : 1000 dilution, Proteintech Inc., Wuhan, China), p-IRS-1 (1 : 1000 dilution, Cell Signaling, USA), p-AktSer473 (1 : 2000 dilution, Abcam, USA), and β-actin (1 : 200 dilution, Boster Biological Technology Co. Ltd., Wuhan, China). Following washing, the membranes were incubated with goat horseradish peroxidase-conjugated secondary antibodies (1 : 50,000 dilution, Boster Biological Technology Co. Ltd., Wuhan, China). The reactions were detected by using an Enhanced Chemiluminescence Western Blot detection kit (Thermo Fisher, USA), and the detected bands were visualized via exposure to an X-ray beam in a dark room.
3
Investigating Hepatoprotective Mechanisms
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LPS and D-GalN were purchased from Sigma. CYG is composed of Herba artemisiae, Salvia miltiorrhiza, and rhubarb and was purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd. OMT was purchased from Shaanxi Baoji Fangsheng Biological Development Co., Ltd., with a purity of >98%. Akt, p-Aktser473, FoxO3a, p-FoxO3a, Bim, Bax, Bcl-2, and active-caspase 3 antibodies were purchased from Abcam Corporation, USA. TRIzol was purchased from Shanghai Pufei Biotechnology Co., Ltd. PCR primers were designed and synthesized by Shanghai Ruiqiang Biotechnology Co., Ltd. The apoptosis detection kit was purchased from Biouniquer, USA.
4
Skeletal Muscle Signaling Pathway Analysis
5
Immunofluorescence Analysis of PI3K/AKT Pathway in FaDu Cells
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After treatment with NP-Ø and NP-427, the FaDu seeded in 24-well plates were fixed with 4% paraformaldehyde (PFA) for 10 min. The cells were blocked for 1 h at 37 °C and incubated overnight at 4 °C with several antibodies: p-AKT-Ser473 (1/75), p-AKT-Thr308 (1/75), and Annexin-V (1/100) from Abcam (Abcam, Cambridge, UK) and p-PDK1-Ser241 and anti-PI3CA (1/100) from Invitrogen (Invitrogen Corporation y Applied Biosystems Inc., Carlsbad, United States). After, the cells were incubated with the secondary antibodies, i.e., Alexa Fluor 546-conjugated goat anti-rabbit antibody (1/250; Molecular Probes, Eugene, United States) or Alexa Fluor 488-conjugated goat anti-rabbit antibody (1/250; Molecular Probes, Eugene, United States). at 37 °C for 45 min. Then, the cells were stained with 300 nM of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, San Luis, CA, United States), reactive with fluorescent blue, which intercalates with DNA, for 5 min at 37 °C. Fluorescence was visualized using an Olympus BX51 microscope. The specificity was assessed by omitting the primary antibody. ImageJ, a free image-processing program, was used for quantitative analysis of the image.
6
Protein Expression Analysis in Cells
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Protein extracts were prepared from cells using RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO). Protein concentration was measured using the bicinchoninic acid method. Equal amounts of proteins were separated by 10% SDS-PAGE and next transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h, followed by incubation with the following primary antibodies: CDK2 (1:1000, Abcam, cat. no. ab206038), PCNA (1:1000, Abcam, cat. no. ab92552), p-Akt Ser473 (1:1000, Abcam, cat. no. ab18206), Akt (1:1000, Abcam, cat. no. ab8805), p-eNOS Ser1177 (1:1000, Cell Signaling Technology, Danvers, MA; cat. no. 9571), eNOS (1:1000, Abcam, cat. no. ab199956), MMP-2 (1:1000, Abcam, cat. no. ab37150), MMP-9 (1:1000, Abcam, cat. no. ab38898), α-SMA (1:1000, Abcam, cat. no. ab204573), and SM22α (1:1000, Arigo, cat. no. ARG63416). The membranes were subsequently cultured with horseradish peroxidase-conjugated goat anti-rabbit IgG. The protein was detected using an enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL) and the band intensity was quantified with Image-Pro Plus 4.5 software. GAPDH served as the internal control.
7
Western Blot Analysis of BMSC Lysates
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All the BMSCs lysates were acquired by adding RIPA lysis buffer, phenylmethylsulfonyl fluoride. Protein concentrations were determined using the BCA Protein Assay kit. We used 10% or 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate approximately 20 μg of protein extracts, and then transferred them to a polyvinylidene fluoride (PVDF) membrane. After blocking the PVDF with milk, we incubated it overnight at 4 °C with the primary antibody. PVDF was washed three times with TBST, and then incubated with goat anti-rabbit antibody for 1 hour, followed by its exposure. Primary antibodies (Rabbit anti Mouse) of RUNX2, p-GSK3βser9, β-catenin were acquired from Cell Signaling Technology, USA; Akt, p-Aktser473, TP63, PTEN, GSK3β from Abcam, UK; β-actin and GAPDH from Cohesion Biosciences, UK. The secondary antibodies (Goat anti Rabbit, HRP) were acquired from Lablead, China.
8
Activin A and TGFβ1 Signaling Analysis
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Activin A was reconstituted in PBS; TGFβ1 in 4 mM HCl according to the manufacturer’s instruction (both R&D, Minneapolis, MN, USA). Final concentrations used were 25 ng/ml and 10 ng/ml, respectively, as previously described [31 (link), 49 (link)–51 (link)]. For inhibition of PI3K, we used LY294002 and for inhibition of MEK1/2, U0126 (both Cell Signaling Technology, Danvers, MA, USA). For immunoprecipitation and Western blotting, we used antibodies against ACVR1B (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ACVR2A (customized by Yenzym, San Francisco, CA, USA), p85 (# 4292), TGFBR1 (# 3712) or TGFBR2 (# 3713, all Cell Signaling), p21 (# sc-65595, Santa Cruz), pan-Akt (# 8805, Abcam, Cambridge, MA, USA), GAPDH (# sc-47724, Santa Cruz), E-Cadherin (# 3195), vimentin (# 3390), pAkt Ser473 (# 4060) and pAkt Thr308 (# 13842, all Cell Signaling). For immunohistochemical analyses, we used p21 (# sc-817, Santa Cruz) ACVR2A (# ab10595), TGFBR2 (# ab78419), pAkt Ser473 (# ab81283, abcam), and pERK1/2 (# ab50011, all Abcam).
9
Immunoblotting Antibody Specifications
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Antibodies used for immunoblots were purchased from the indicated companies: Catalase (1:3000 dilution, #ab1877) (Abcam), p-Akt (Ser473) (1:2000 dilution, #9272), Akt (1:2000 dilution, #9271), p-eNOS (Ser1177) (1:1000 dilution, #9571), eNOS (1:1000 dilution, #9586), p-FoxO1 (Thr24, Ser256) (1:1000 dilution, #9464 and #9461), FoxO3 (1:1000 dilution, #9467), GAPDH (1:3000 dilution, #2118), Histone H3 (1:500 dilution, #9715), Lamin A/C (1:5000 dilution, #4777), p-Mst1 (Thr183) (1:1000 dilution, #3681), p-YAP (Ser127) (1:1000 dilution, #4911), YAP (1:2000 dilution, #4912) (Cell Signaling Technology), FoxO1 (1:2000 dilution, #1874-1) (epitomics), α-tubulin (1:5000 dilution, #T-6199), FLAG (1:2000 dilution, #F3165) (Sigma), MnSOD (1:3000 dilution, #611580), Mst1 (1:2000 dilution, #611052) (BD Biosciences), and Lats2 (1:1000 dilution, #ab54073 and #A300-479A) (Abcam and Bethyl Laboratories). The p-Lats2 (S872 and T1041) (1:500 dilution) antibodies were generated as described41 (link).
10
CXCL5/CXCR2 Signaling in Prostate Cancer
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The primary antibodies against CXCL5, CXCR2, phosphorylated (p)-STAT3 (Tyr705), STAT3, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-AKT (Ser473), AKT, and β-actin were purchased from Abcam. The human Penl1, Penl2, 149RCa, and LM156 PC cell lines were kindly provided by Prof. Hui Han (Department of Urology, Cancer Hospital, Sun Yat-Sen University) [23 (link)]. The cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum as previously described [23 (link)]. Lentiviral short hairpin (sh)RNAs targeting shCXCL5 or shCXCR2 were purchased from GeneCopoeia Inc, and were used as previously described [24 (link),25 (link)].
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