Protein lysates were prepared in RIPA buffer and quantified using Bradford Assay (Bio-Rad, #5000001). SDS-PAGE and Western blots were performed according to standard procedures using antibodies against the cell cycle regulators c-Myc (Cell Signaling, #5605), p21 (Santa Cruz Biotechnology, #SC-756) and Sirt1 (Abcam, #ab110304); the differentiation markers CDH5 (Cell Signaling, #2158 S), PECAM1 (Cell Signaling, #3528 S), α-SMA (Sigma, #A2547) and SM22-α (Abcam, #ab14106), the kinases GSK-3β (Cell Signaling, #9832 S), AKT (Cell Signaling, #9272) and phosphorylated forms phospho-GSK-3β (Ser9) (Cell Signaling, #5558 S) and phospho-AKT (Ser473) (Cell Signaling, #9271), and endogenous controls Actin (Sigma-Aldrich, #A2066), Gapdh (Cell Signaling, #5174) and Tubulin (Cell Signaling, #2146). Prior to blocking and antibody incubation, membranes were stained with Ponceau S reagent (Sigma-Aldrich, #P3504) to visualize protein bands and cut at specific sizes so different antibodies could be tested at the same time. Densitometry analysis of western blots was performed using Quantity One software (Bio-Rad Laboratories).
Phospho akt ser
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-AKT (Ser) is a laboratory reagent that detects the phosphorylation of the AKT protein at the serine residue. This phosphorylation event is an important signaling mechanism that regulates various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
α sma, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Pecam 1, by Cell Signaling Technology (1 mentions)
Ponceau s reagent, by Merck Group (1 mentions)
Sirt1, by Abcam (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Sm22α, by Abcam (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Eif4ebp1, by Cell Signaling Technology (1 mentions)
G6pdh, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phospho ulk1, by Cell Signaling Technology (1 mentions)
Phospho rps6kb thr389, by Cell Signaling Technology (1 mentions)
Phospho eif4ebp1, by Cell Signaling Technology (1 mentions)
Phospho rps6, by Cell Signaling Technology (1 mentions)
Gapdh antibody, by Beyotime (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
5 protocols using phospho akt ser
1
Western Blot Analysis of Cell Signaling
2
Western Blot Antibody Panel for Cell Signaling
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Primary antibodies against GFP (Takara Bio, 632380, AB_10013427; 1:5000), G-6-PDH (Sigma-Aldrich, A9521, AB_258454; 1:5000), Adh1 (Millipore, 126745, AB_564196; 1:200000), RPS6 (Cell Signaling Technology, 2217, AB_331355; 1:1000), phospho-RPS6 (Ser235-236) (Cell Signaling Technology, 4856, AB_2181037; 1:1000), RPS6KB (Cell Signaling Technology, 2708, AB_390722; 1:1000), phospho-RPS6KB (Thr389) (Cell Signaling Technology, 9205, AB_330944; 1:1000), EIF4EBP1 (Cell Signaling Technology, 9452, AB_331692; 1:1000), phospho-EIF4EBP1 (Thr37/46) (Cell Signaling Technology, 2855, AB_560835; 1:1000), phospho-EIF4EBP1 (Ser65) (Cell Signaling Technology, 9451, AB_330947; 1:1000), AKT1 (Cell Signaling Technology, 4691, AB_915783; 1:1000), phospho-AKT(Ser473) (Cell Signaling Technology, 4060, AB_2315049; 1:1000), phospho-AKT(Thr308) (Cell Signaling Technology, 13038, AB_2629447; 1:1000), ULK1 (Cell Signaling Technology, 8359, AB_11178668; 1:1000), phospho-ULK1(Ser757) (Cell Signaling Technology, 6888, AB_10829226; 1:1000), MAP1LC3 AB (Cell Signaling Technology, 12741, AB_2617131; 1:1000), β-actin (Cell Signaling Technology, 4967, AB_330288; 1:1000) were used.
3
Western Blot and qRT-PCR Analysis of Signaling Pathways
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The protein samples from stimulated or transfected cells were extracted by using the Laemmli Sample Buffer (Bio-Rad Laboratories, Inc., USA). The concentrations of protein samples were quantified by using the BCA™ Protein Assay Kit (Beyotime #P0012). Antibodies purchased from Cell Signaling Technology (Beverly, MA) included: phospho-STAT3 (Tyr705) (#9145), phospho-STAT3 (Ser727) (#49081), STAT3 (#4904), phospho-JAK2 (Tyr1007/1008) (#3771), JAK2 (#3230), phospho-JAK1 (Tyr1034/1035) (#74129), JAK1 (#3344), phospho-TYK2 (Tyr1054/1055) (#68790), TYK2 (#14193), phospho-STAT1 (Tyr701) (#7649), STAT1 (#14994), phospho-AKT (Ser473) (#4060), AKT(Pan) (#4821), phospho-NF-κB p65 (Ser536) (#3033), NF-κB p65 (#8242), cyclin D1 (#2978), cyclin A2 (#4656), cyclin B1 (#12231), Caspase-3 (#29629), Bcl-xL (#2762), and Survivin (#2803). PARP antibody and GAPDH antibody were purchased from Beyotime (#AP102) and Genscript (#A00192), respectively. WB analysis was conducted as previously described58 (link). Three independent experiments were performed using samples collected at different days. qRT-PCR was conducted as described previously59 (link). All the primers used were listed in Fig. S1b .
4
Protein extraction and analysis protocol
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Radioimmunoprecipitation assay (RIPA) buffer (Solarbio, China, #R0010) mixed with protease and phosphatase inhibitors was used to isolate total proteins from cells. The protein concentration was measured using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, #A53226), according to the manufacturer's instructions. Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): PI3K110α (C73F8), β-Tubulin, phospho-Akt (Ser 473), phosphor-S6 (Ser 235/236), PKCα, PARP (46D11), Cleaved PARP (Asp 214), Cleaved Caspase 3 (Asp 175), NRF2 (D1Z9C) and SLC7A11 (D2M7A). Horseradish peroxidase (HRP)-labeled goat anti-rabbit/mouse IgG (1:3000 dilution, ZSGB-BIO, Beijing, China, #ZB2301) were used as secondary antibodies. Enhanced chemiluminescence (ECL) substrate (CWBIO, Beijing, China, #CW00495). FluorChem E System (Protein Simple, USA) was used to evaluate protein expression. Image J software was used to quantify the level of proteins.
5
Western Blot Analysis of Cell Signaling Proteins
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Protein expression was investigated by western blot analysis using 10–40 μg of total protein. Tissue was homogenized, cells were pelleted. Cell lysis was performed (Cell lysis buffer, Cell Signaling Technology, Danvers, MA, USA) and protein electrophoresis initiated. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% milk in TBS-Tween, and probed overnight at 4°C with the following primary antibodies: SMAD7 (rabbit anti-mouse, Invitrogen, USA), PTEN (rabbit anti-mouse, Abcam), Foxo3a (rabbit anti-mouse, Cell signaling Technology), phospho-AKT (ser) (rabbit anti-mouse, Cell signaling Technology), AKT (rabbit anti-mouse, Cell signaling Technology), phospho-ERK (44/42) (rabbit anti-mouse, Cell signaling Technology), ERK (rabbit anti-mouse, Cell signaling Technology). Antibody binding was visualized by chemiluminescence (Super-Signal West Pico Chemiluminescent, Thermo Scientific, Rockford, IL, USA). Rabbit anti-mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma-Aldrich) was used as an internal loading control and for normalization of protein quantification. Immunoblots were scanned and quantified using ImageJ densitometry software. Recombinant OPN was purchased at RnD systems (USA). The phosphoinositide-3-kinase (PI3-kinase) inhibitor wortmannin was obtained from Sigma-Aldrich.
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