Cells were washed and resuspended in 2× SDS sample buffer at 106 cells/100 µl and boiled for 5 min. Samples were separated on Novex 4–12% Tris-Glycine gel (Invitrogen) and transferred to a PVDF membrane (Immobilon-P; EMD Millipore). Proteins were detected with the following primary antibodies: p-AKT(S473) (D9E) and AKT (5G3; Cell Signaling Technology). An anti–rabbit IgG-HRP antibody (GE Healthcare) was used as the secondary antibody.
P akt s473 d9e
Manufactured by Cell Signaling Technology
P-AKT(S473) (D9E) is a lab equipment product from Cell Signaling Technology. It is an antibody that detects phosphorylation of AKT at serine 473.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg hrp antibody, by GE Healthcare (1 mentions)
Novex 4 12 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Odyssey 2800, by LI COR (1 mentions)
Erk1 2 9102, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Ha mms 101r, by Fortrea (1 mentions)
P mtors2448, by Cell Signaling Technology (1 mentions)
Gsk3α β, by Cell Signaling Technology (1 mentions)
Ppdk1 s241, by Cell Signaling Technology (1 mentions)
P70 s6 kinase 49d7, by Cell Signaling Technology (1 mentions)
Sc 1616, by Santa Cruz Biotechnology (1 mentions)
4 protocols using p akt s473 d9e
1
Western Blot Analysis of p-AKT and AKT
2
Proteomic Analysis of Cell Signaling
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Cells were processed according to the previously described protocol [52 (link)] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance). Proteins were detected by horseradish peroxidase-linked secondary antibodies (Cell Signaling, distributed by Euroclone, Milan, Italy) and revealed using the West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Life Technology, Milan, Italy). Chemiluminescent signal was revealed on Amersham high performance chemiluminescence films (Hyperfilms Amersham, distributed by Biogenerica, Catania, Italy), or using the LI-COR Odyssey 2800 (Li-COR Inc., Lincoln, NE, USA) and the software ImageStudioLite (version 5.2). β-actin (Sigma Aldrich, Merck, Milan, Italy) served as loading control. Original western blots and densitometric analyses of all blots shown are reported in Figures S3–S8 .
3
Antibody-based Signaling Pathway Analysis
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Antibodies of COL3A1 (13548-1-AP), cyclin D1 (60186-1-Ig) and PI3K (20583-1-AP) were purchased from Proteintech (Wuhan, Hubei, China). GAPDH antibody (CW0100) was bought from CWBIO, Beijing, China. Other antibodies were purchased from Cell Signaling Technology (CST): PDK1 (#3062), p-PDK1S241 (#3061), p-AktS473 (D9E, #4060), p-AktT308 (244F9, 4056), p-p70S6KT389 (108D2, #9234), p70 S6 Kinase (49D7, #2708), p-mTORS2448 (#2971), mTOR (#2972), p-GSK-3α/βS21/9 (D17D2, #8566) and GSK-3α/β (D75D3,#5676).
4
Western Blot Analysis of Oncogene Pathways
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Anti-KRAS (ab55391) antibodies were purchased from Abcam (Cambridge, MA). Anti-NRAS (C-20, sc-519) and Anti-MYC (N-262, sc-764) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAPK1, –MEK1/2 (L38C12), –p-MEK1/2 (S217/221,41G9), AKT and p-AKT (S473, D9E) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Equivalent gel loading was confirmed by probing with antibodies against actin (sc-1616; Santa Cruz) or calnexin (C5C9; Cell Signaling). The intensity of the resulting bands was measured by ImageJ 1.48 s (http://imagej.nih.gov/ij ). The intensity ratio of each band respective to the corresponding actin intensity was used for relative quantification.
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