Cell-lysate protein concentrations were determined using a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes, which were probed with primary antibodies: mouse polyclonal Akt, GSK-3β, CDK4, and β-catenin (1:1000; Bioss, Beijing, China); rabbit polyclonal p-Akt, vimentin, cyclin D1, MYH9, c-Jun, vimentin, Sox2, and Oct-4 (1:1000; Bioss, Beijing, China), and rabbit polyclonal PI3K, p-PI3K, and HMGA1 (1:1000; Cell Signaling Technology, Danvers, USA). Rabbit monoclonal anti-β-actin antibody (1:1000; CoWin Bioscience, Beijing, China) was used for normalization. HRP-conjugated anti-rabbit or anti-mouse IgG antibodies were used as secondary antibodies (1:2000; CoWin Bioscience, Beijing, China). Proteins were detected using an enhanced chemiluminescence reagent (Thermo Scientific, Waltham, MA, USA). Images were captured using a ChemiDocTM CRS + Molecular Imager (Bio-Rad, Hercules, CA, USA). Grayscale semi-quantification of the bands is shown in Supplemental Fig. 4 for all western blots.
P akt
Manufactured by Bioss Antibodies
Sourced in China, United States
P-AKT is a lab equipment product that measures the phosphorylation of the AKT protein. AKT is a key regulator of cellular processes, and its phosphorylation status is an important indicator of cellular signaling activity.
Automatically generated - may contain errors
Lab products found in correlation
P pi3k, by Bioss Antibodies (3 mentions)
Cyclin d1, by Bioss Antibodies (2 mentions)
P pi3k, by Cell Signaling Technology (2 mentions)
β actin, by Bioss Antibodies (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Bioss Antibodies (1 mentions)
Chemidoc crs molecular imager, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
C jun, by Bioss Antibodies (1 mentions)
Hmga1, by Cell Signaling Technology (1 mentions)
β catenin, by Bioss Antibodies (1 mentions)
Gsk3β, by Bioss Antibodies (1 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Mem medium, by Thermo Fisher Scientific (1 mentions)
Du145, by Thermo Fisher Scientific (1 mentions)
Mk2206, by MedChemExpress (1 mentions)
Ab6721, by Abcam (1 mentions)
Minimum essential medium (mem), by Lonza (1 mentions)
Goat anti rabbit igg hrp, by Abcam (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
14 protocols using p akt
1
Western Blot Protein Analysis
2
Prostate Cancer Cell Culture Protocols
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Human prostate cancer cells PC-3 and DU 145 were purchased from the Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Sciences (Shanghai, China) and C4-2 were obtained from ATCC (Manassas, VA, USA). PC-3 cells were cultured in F-12 medium (Gibco, Rockville, IN, USA), DU 145 were kept in MEM medium (Gibco, Rockville, IN, USA) and C4-2 were maintained in 400 mL MEM (Lonza, Basel, Switzerland) plus 100 mL F12 Medium. All the medium was supplemented with 10% fetal bovine serum (FBS) and all cultured medium changed every 3 days. All cells were maintained at 37˚C with 5% CO2 and 95% humidity.
Antibodies used in our study were PSMC2 (Cat # SC-166972, Santa Cruz, CA, USA), GAPDH Rabbit (Cat #AP0063, Bioworld, St. Louis, MN, USA), HRP goat anti-rabbit/mouse IgG (Cat # A0208/ A0216, Beyotime, Beijing, China), Akt and Cyclin D1 (Cat #4685/2978, CST, Danvers, MA, USA), p-Akt (Cat #bs-5193r, Bioss, Beijing, China), CDK6 (Cat #ab151247, Abcam, Cambridge, MA, USA), P21 (Cat #BM3990, Boster, Wuhan, China), Ki67 (Cat #ab16667, Abcam, Cambridge, MA, USA) and HRP goat anti-rabbit IgG (Cat #ab151247, ab40776, ab76125, ab16667 and ab6721, Abcam, Cambridge, MA, USA).
Akt inhibitor MK-2206 was purchased from MedChemExpress (Cat #HY-10358).
Antibodies used in our study were PSMC2 (Cat # SC-166972, Santa Cruz, CA, USA), GAPDH Rabbit (Cat #AP0063, Bioworld, St. Louis, MN, USA), HRP goat anti-rabbit/mouse IgG (Cat # A0208/ A0216, Beyotime, Beijing, China), Akt and Cyclin D1 (Cat #4685/2978, CST, Danvers, MA, USA), p-Akt (Cat #bs-5193r, Bioss, Beijing, China), CDK6 (Cat #ab151247, Abcam, Cambridge, MA, USA), P21 (Cat #BM3990, Boster, Wuhan, China), Ki67 (Cat #ab16667, Abcam, Cambridge, MA, USA) and HRP goat anti-rabbit IgG (Cat #ab151247, ab40776, ab76125, ab16667 and ab6721, Abcam, Cambridge, MA, USA).
Akt inhibitor MK-2206 was purchased from MedChemExpress (Cat #HY-10358).
3
Gastric Cancer Drug Resistance Model
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The human gastric cancer cell line BGC823 was purchased from KeyGEN BioTECH Co., Ltd. (China).
The drug-resistant cell line BGC823/5-Fu was established by the low-dose multiple shock method in our previous research [17 (link)] and stored at -196 °C. The drug resistance index of BGC823/5-Fu was 13.
The serum-free medium (SFM) was composed of 95% DMEM/F12 (Gibco, USA), 1% 2 µg/ml EGF (Peprotech, USA), 1% 2 µg/ml bFGF (Peprotech, USA), 2% B27 (Gibco, USA), 1% 100 U/ml penicillin/streptomycin (Gibco, USA) and 0.4 U insulin (Sigma, USA). The IGF-1 was purchased from Sigma (USA). The P-gp, MRP1, PI3K, p-PI3K, AKT, p-AKT, Nrf2 and β-actin antibodies were purchased from Bioss, Inc. (China) and Proteintech Group, Inc. (USA). HRP-labeled Goat Anti-Rabbit IgG, HRP-labeled Goat Anti-Mouse IgG and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG were purchased from Beyotime Biotechnology (China). The Annexin V-FITC/PI Kit was purchased from KeyGEN BioTECH Co., Ltd. (China). CD44 MicroBead Kit was purchased from Miltenyi Biontec. (Germany). Lipofectamine2000 was purchased from Invitrogen (USA).
The drug-resistant cell line BGC823/5-Fu was established by the low-dose multiple shock method in our previous research [17 (link)] and stored at -196 °C. The drug resistance index of BGC823/5-Fu was 13.
The serum-free medium (SFM) was composed of 95% DMEM/F12 (Gibco, USA), 1% 2 µg/ml EGF (Peprotech, USA), 1% 2 µg/ml bFGF (Peprotech, USA), 2% B27 (Gibco, USA), 1% 100 U/ml penicillin/streptomycin (Gibco, USA) and 0.4 U insulin (Sigma, USA). The IGF-1 was purchased from Sigma (USA). The P-gp, MRP1, PI3K, p-PI3K, AKT, p-AKT, Nrf2 and β-actin antibodies were purchased from Bioss, Inc. (China) and Proteintech Group, Inc. (USA). HRP-labeled Goat Anti-Rabbit IgG, HRP-labeled Goat Anti-Mouse IgG and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG were purchased from Beyotime Biotechnology (China). The Annexin V-FITC/PI Kit was purchased from KeyGEN BioTECH Co., Ltd. (China). CD44 MicroBead Kit was purchased from Miltenyi Biontec. (Germany). Lipofectamine2000 was purchased from Invitrogen (USA).
4
Western Blot Analysis of Protein Expression
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Cells were lysed in protein lysis buffer and the proteins were subjected to quantity determination. Then the proteins were resolved on SDS-PAGE. The proteins in denaturing acrylamide gels were transferred to PVDF membrane and non-specific antigens were blocked by 5% nonfat milk. Primary antibodies for RBP2 (Abcam USA), β-actin (Sigma, USA), Sp1 (Cell Signaling, USA), CagA (Abcam USA), Cyclin D1 (Bioss, China), AKT (Bioss, China) and p-AKT (Bioss, China) were used to incubate overnight at 4°C and then immunoblot detection was performed with chemiluminescence (Millipore, USA) according to the protocol.
5
Investigating Anti-inflammatory Mechanisms of Traditional Chinese Medicines
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EB and OT were provided by Hangzhou Animal Medicine Factory (Hangzhou, China). TongJingBaoKeLi (TJB, SFDA approval number: Z41021972) was bought from Zhongjing Wanxi Pharmaceutical Co., Ltd. (Henan, China). ELISA Kits for prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), thromboxane B2 (TXB2), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), nitric oxide (NO), and cyclooxygenase-2 (COX-2) were supplied by Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China), and the Ca2+ kit was bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), p-PI3K and p-AKT were obtained from Bioss (Beijing, China). All other reagents were analytical grade, and water was ultrapure water.
6
Comprehensive Protein Analysis via Western Blot
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Total protein was extracted using RIPA buffer containing proteinase inhibitor (Best Biological, Jiangsu, China). Western blot was conducted as previously described [12 (link)]. The primary antibodies for P-ERK1, ERK1, NF-κB1, RELA, P-mTOR, mTOR, VEGFA, PD-L1, P-P13K, P13K, P-AKT, AKT, TNF-α, P-EGFR, EGFR and HIF-1α were purchased from Bioss (Beijing, China). Total protein level was normalized to β-actin.
7
Liver Development Immunohistochemistry Protocol
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In brief, the liver specimens from E12.5, E18.5 and PND1 were isolated for preparing paraffin block before performing microtome section. Subsequently, 5 μm paraffin-embedded sections were subjected to deparaffinization and gradient dehydration. After being blocked with fresh bovine serum albumin buffer (5%, v/v), the dewaxed sections were hatched with primary antibodies against Cyp4a, p-AKT, mTOR, IRβ, IGF-1R, FGF21, CYP3A4, and NOS2 (1:150–200 dilutions; Bioss, Beijing, China) at 4°C overnight. After being washed with neutral phosphate buffered saline buffer, the sections were re-hatched with secondary antibody-coupled fluorescent dye (Beyotime Biotechnology, China) under light-free conditions for approximately 1 h at indoor temperature. Subsequently, 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) dye was used for nuclear staining, in which the positive cells were identified under different magnification view and counted for statistic determination, as previously reported [30 (link)].
8
Western Blot Analysis of AKT/PI3K Pathway
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Wound tissues were lysed with RIPA buffer and total protein was extracted and quantified using the BCA kit (Thermo, USA). After protein denaturation at 100 °C for 10 min, an equal volume of loading buffer was added to the sample before SDS-PAGE electrophoresis. Proteins were then transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 1 h with gently shaking. Membranes were incubated with the following primary antibodies; AKT (1:1000, 4691S, Cell Signaling Technology), p-AKT (1:300, bs-0876R, Bioss), PI3K (1:500, 4292S, Cell Signaling Technology), P-PI3K (1:300,17366S, Cell Signaling Technology), or β-actin (1:5000, G1001, GXY bioSci & Tech Co., Ltd.) overnight at 4 °C. Afterward, Anti-Rabbit IgG (H + L) Antibody, DyLight™ 680-Labeled (1:5000, 5230-0402, SeraCare) was incubated at room temperature for 1 h. Protein bands were visualized using the far-infrared laser imaging system (LI-COR, USA). Gray value intensities of the bands were quantified using the Image J software.
9
Western Blot Analysis of Exosomal Proteins
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Exosomes or cells were lysed with RIPA buffer, then the protein concentration was measured using the BCA protein detection kit (Beyotime, China). Each sample was mixed with protein loading buffer (5×) and heated at 95°C for 10 min to denature. Then protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Germany). PVDF membranes were blocked with 5% skim milk for 1 h, and primary antibody (MMP-13, collagenase II, IGFR1, p-PI3K, p-AKT, GAPDH, and β-ACTIN) (Bioss, China) was added in for incubating overnight at 4°C, then the secondary antibody conjugated with horseradish peroxidase was incubated for 2 h at room temperature. Fluorescence was analyzed using the enhanced chemiluminescence kit (Tanon, China) and imaged by the luminescent image analyzer (Tanon, China), and the absorbance value of each band was calculated using the ImageJ software.
10
Evaluation of Molecular Pathways
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CocoaVia® was purchased from Mars Inc., (Hackettstown, NJ, USA). Primary antibodies Nitro-tyrosine, IL-6, NFkB2, P-Erk, P-akt antibody were obtained from Bioss antibodies, Novusbio, and Cell Signaling Technology (Danvers, MA USA). In Situ Cell Death Detection Kit, AP was from Roche (Basel, Switzerland).
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