Western blotting was performed as described previously, [16 (link)] using the following primary antibodies: HNRNPLL (26769-1-AP, Proteintech, USA), MYO1B (sc-393,053, Santa Cruz Biotechnology, USA), FLAG (14,793 S, Cell Signaling Technology, USA), phospho-AKT (4060, Cell Signaling Technology), phospho-mTOR (5536T, Cell Signaling Technology), and β-actin (ab6276, Abcam, UK). Relative protein levels were analyzed by ImageJ.
Phospho akt 4060
Manufactured by Cell Signaling Technology
Sourced in United States
The Phospho-AKT (4060) is a primary antibody that detects endogenous levels of AKT protein when phosphorylated at Serine 473. It is designed for use in western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (1 mentions)
Anti actin actn05 c4, by Abcam (1 mentions)
Irdye 800cw goat anti rabbit 926 32 211, by LI COR (1 mentions)
Akt 9272, by Cell Signaling Technology (1 mentions)
Foxo3a 2497, by Cell Signaling Technology (1 mentions)
Foxo1 2880, by Cell Signaling Technology (1 mentions)
Resatorvid tak 242, by MedChemExpress (1 mentions)
Cleaved caspase 3 9661, by Cell Signaling Technology (1 mentions)
β actin antibody, by Merck Group (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Phospho p38 9215, by Cell Signaling Technology (1 mentions)
Tlr4 antibody, by Santa Cruz Biotechnology (1 mentions)
Ab48824, by Abcam (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
Actin nb600 501, by Novus Biologicals (1 mentions)
X ray film, by AGFA HealthCare (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Luminol reagent, by GE Healthcare (1 mentions)
Acetone, by Merck Group (1 mentions)
8 protocols using phospho akt 4060
1
Western Blotting for Protein Analysis
2
Antibody Characterization for LRIG1 Analysis
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The antigen peptides for all LRIG1 antibodies used are indicated in Supplementary Fig. 1B . For immunohistochemistry: Anti-LRIG1 antibody (Atlas Antibodies AB, Bromma, Sweden, catalog no. HPA011846), 4.0 mg/ml. The 1A8 anti-LRIG1 antibody is described in the Supplementary Materials. For western blots: EGFR #2232, 1:1000; Phospho-p44/42 MAPK #4370, 1:1000; Phospho-AKT #4060, 1:1000 (Cell Signaling Technology Inc, Danvers, USA); Anti-Actin #ACTN05/C4, 1:3000 (Abcam, Cambridge, UK). Anti-LRIG1 Vina [49 (link)], 1:1000 (Agrisera AB, Vännäs, Sweden). Secondary antibodies: IRDye® 680RD Donkey anti-Mouse #926-68072, 1:15000; IRDye® 800CW Goat anti-Rabbit #926-32211, 1:15000 (LI-COR, Lincoln, NE, USA).
3
Western Blot Analysis of Foxo and Akt
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Cells were washed in PBS and then lysed. The protein concentration of each cell lysate was determined and equated. Prepared samples were boiled in sample buffer at 100 °C for 5 minutes. Samples were loaded in SDS-PAGE gel and the gel ran at 140–160 V. After electrophoresis proteins were transferred to the PVDF membrane. Membranes were blocked for 1.5 h at room temperature using blocking buffer (5% nonfat dry milk) and incubated with primary antibodies for 1 h at room temperature or overnight at +4 °C. Then membranes were incubated with HRP-conjugated secondary antibodies diluted in blocking buffer for 1h at room temperature. As primary antibodies, we used antibodies to Foxo1 #2880, Foxo3a #2497, phospho-Foxo3a #9466, Akt #9272, phospho-Akt #4060 (Cell Signalling).
4
Resatorvid (TAK-242) Antiinflammatory Mechanism
5
Insulin Signaling Pathway Analysis
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In Western blot analyses, 30–40 μg of protein was used to probe for polymerase I and transcript release factor (PTRF; ab48824; Abcam); the ratio of phospho-caveolin-1 (3251) to total caveolin-1 (3267, clathrin (4796), insulin receptor (3020), ratio of phospho-hormone–sensitive lipase (Hsl;4139) to total Hsl (4107), ratio of phospho-Akt (4060) to total Akt (9272), and ratio of phospho-p44/42 MAPK (4370) to total p44/42 MAPK (4695; all from Cell Signaling Technology); and lipoprotein lipase (Lpl; sc-32885; Santa Cruz Biotechnology, Dallas, TX, USA) in gonadal tissue from mice that were left unfed overnight and during a 15-min insulin-stimulation (2 U/kg body weight). Actin (NB600-501; Novusbio, Littleton, CO, USA) and vinculin (13901; Cell Signaling Technology) were used as loading references.
6
Western Blot Analysis of Cellular Proteins
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Cells were lysed with lysis buffer (Cell Signaling Technology, Danvers, MA, USA), and equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8–12% gels and transferred to nitrocellulose membranes. The membranes were blocked by incubating with 5% non-fat dry milk in PBS containing 0.2% Tween-20 (PBST) for 1 h, and then incubated with primary antibodies overnight at 4 °C. After three washes with PBST, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse (#A120-101P) or rabbit (#A90-116P) (Bethyl Laboratories, Inc., Montgomery, TX, USA) for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotechnology, Amersham, UK) and X ray film (AGFA, Mortsel, Belgium). Primary antibodies against ARPP19 (#MBS9609642) were obtained from MyBioSource (San Diego, CA, USA); phospho-AKT (#4060) and AKT (#4691) were obtained from Cell Signaling Technology; c-Myc (#sc-764), cyclin D1 (#sc-753), E-cadherin (#sc-8426), and β-actin (#sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
7
Western Blot Analysis of Cellular Proteins
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Western blot was performed as described previously [51 (link)]. Briefly, membranes were incubated with primary antibody at 4°C overnight, followed by incubation with secondary antibody at room temperature for 1 hour. Immunoreactive bands were detected using Western blot Luminol reagent (GE Healthcare, Waukesha, WI). Antibodies used were CHD5 (23320002, Novus), EZH2 (18-7395, Invitrogen), MDM2 (sc-813, Santa Cruz, CA), MYC (sc-764, Santa Curz, CA), cleaved poly (ADP-ribose) polymerase (9541, Cell Signal), phospho-AKT (4060, Cell Signal), α-tubulin (MS-581, Thermo Lab Vision, MI); p53 (M7001), anti-mouse IgG-HRP (P0161), anti-rabbit IgG-HRP (P0448) (Dako, Glostrup, Denmark).
8
Evaluating RAGE and HDAC Inhibitors in Toluene Diisocyanate Exposure
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TDI (toluene-2, 4-diisocyanate, ≥ 98.0%), acetone, and methacholine were obtained from Sigma-Aldrich (Shanghai, China). The vehicle that TDI was dissolved in was a mixture of acetone and olive oil (AOO): the ratio of acetone to olive oil was 2:3 for sensitization, and 1:4 for challenge. FPS-ZM1 (RAGE inhibitor), JNJ-26482585 (HDAC inhibitor), romidepsin (HDAC inhibitor), and MK2206 (AKT inhibitor) were purchased from Selleck (SelleckChem, Shanghai, China). JNJ-26482585 is a novel second-generation HDACi with highest potency for HDAC1 and romidepsin is a selective inhibitor of HDAC1 and HDAC2.
Anti-RAGE (ab37647), and anti-HDAC1 (ab109411) were from Abcam. Anti-total-AKT (#4691 ) and Phospho-Akt ( #4060 ) were purchased from Cell Signaling Technology (Boston, MA, USA).
Anti-RAGE (ab37647), and anti-HDAC1 (ab109411) were from Abcam. Anti-total-AKT (#4691 ) and Phospho-Akt ( #4060 ) were purchased from Cell Signaling Technology (Boston, MA, USA).
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