For whole cell protein extracts, cells were collected and lysed with RIPA buffer (Boston Bioproducts, Ashland, MA). Protein was quantitated using the BCA assay (Pierce, Rockford, IL). Lysates were electrophoretically separated using 12% Tris HCl gels (Biorad, Hercules, CA) and transferred to nitrocellulose membranes. Western blotting was performed using antibodies specific for the following proteins: pSTAT5, STAT5, pAkt, Akt, p56lck, phosphor-p56lck, pErk1/2, Erk1/2, pFoxO1/FoxO3a, FoxO1, GATA-1 (all from Cell Signaling Technologies, Danvers, MA), NFATc1, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP. Average densitometric ratio was calculated for each blot by normalizing to an actin loading control using Image J software.
Pfoxo1 foxo3a
Manufactured by Cell Signaling Technology
Sourced in United States
PFoxO1/FoxO3a is a rabbit monoclonal antibody that specifically recognizes phosphorylated forms of FoxO1 and FoxO3a proteins. FoxO1 and FoxO3a are transcription factors that play key roles in cellular processes such as apoptosis, cell cycle regulation, and glucose metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Foxo1, by Cell Signaling Technology (2 mentions)
Goat anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P stat5, by Cell Signaling Technology (1 mentions)
Stat5, by Cell Signaling Technology (1 mentions)
Tris hcl gel, by Bio-Rad (1 mentions)
Gata1, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Nfatc1, by Cell Signaling Technology (1 mentions)
Pras40, by Cell Signaling Technology (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Gsk3α β, by Cell Signaling Technology (1 mentions)
P bad, by Cell Signaling Technology (1 mentions)
Pakt t308, by Cell Signaling Technology (1 mentions)
Pgsk3α β, by Cell Signaling Technology (1 mentions)
Foxo3a, by Cell Signaling Technology (1 mentions)
Pakt s473, by Cell Signaling Technology (1 mentions)
3 protocols using pfoxo1 foxo3a
1
Whole Cell Protein Extraction and Western Blotting
2
Western Blot Analysis of AKT Signaling
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Western analyses were performed as described25 (link). For each experiment, lysates from a cell line grown with or without serum were run on the same gel and all gels were run at the same time. Two gels were transferred to a single membrane. If more than one membrane was used for an experiment, a single solution for each primary and secondary antibody pair was made and divided between the two membranes that were subsequently hybridized in separate trays. Antibodies used included pAKT(S473) (product #4060), pAKT(T308) (product #2965); pan-AKT (product #2920 and 4691), pPRAS40 (product #2997), PRAS40 (product # 2610), pS6(S235) (product #2211), pS6(S240) (product #5364) S6 (product #2317 and 2217), pFOXO1/FOXO3a (product #9464), FOXO3a (product #2497), pGSK3α/β (product #9331), GSK3α/β (product #5676), pBAD (product #4366) and BAD (product #9239) from Cell Signaling Technologies (Danvers, MA, USA) or β-actin (product #A5441) from Sigma-Aldrich (St. Louis, MO, USA). Membranes were scanned and intensities calculated using a semi-automated infrared imaging system (Odyssey scanner and Image Studio Lite software, LiCor BioSciences Inc., Lincoln, NE, USA). Histograms show ratios of infrared signal intensities of indicated antibodies. For experiments using two membranes, histograms of infrared signal ratios graphed by membrane are shown in Supplementary Fig. 8 .
3
Bafilomycin A1 and Dorsomorphin Dihydrochloride Protocol
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Bafilomycin A1 (Baf A1) was obtained from MedChem Express (catalog no. HY‐100558, Princeton, USA). Dorsomorphin dihydrochloride (Compound C dihydrochloride) was purchased from MedChem Express (catalog no. HY‐13418, Princeton, USA). Antibodies against p‐AMPK (1:1000; catalog no. AF3423), AMPK (1:1000; catalog no. DF6361), TFEB (1:1000; catalog no. AF7015), IL‐6 (1:1000; catalog no. DF6087) and H3 (1:3000; catalog no. BF9211) were purchased from Affinity Biosciences (OH, USA). LC3A/B (1:1000; catalog no. 4108), IL‐1β (1:1000; catalog no. 12242), GAPDH (1:1000; catalog no. 5174), p‐FOXO1/FoxO3a (1:1000; catalog no. 9464) and FOXO1 (1:1000; catalog no. 2880) were purchased from Cell Signaling Technology (Danvers, MA, USA). LAMP1 (1:1000; catalog no. A16894), ABCA1 (1:1000; catalog no. A16337), IL‐18 (1:1000; catalog no. A16737), Beclin 1 (1:1000; catalog no. A11761), CD36 (1:1000; catalog no. A5792), TNF‐α (1:1000; catalog no. A11534) and CTSD (1:1000; catalog no. A13292) were purchased from ABclonal Technology (Wuhan, China). SQSTM 1/p62 (1:1000; catalog no. ab240635) was purchased from Abcam (Cambridge, the United Kingdom). The secondary antibodies HRP‐conjugated anti‐mouse IgG and HRP‐conjugated anti‐rabbit IgG were purchased from Affinity Biosciences (OH, USA).
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