Proteins were extracted from tissues by homogenizing in RIPA buffer containing protease inhibitors. Antibodies recognizing total AKT(4691S; 1:1000; 10% acrylamide gels), phospho-AKT (Ser473; 4060S; 1:1000, 10% acrylamide gels), and cytochrome oxidase (COX)-IV (4844S; 1:1000, 15% acrylamide gels) were purchased from Cell Signaling, and actin (A-4700; 1/1000) from Sigma-Aldrich. Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. Following detection of phospho-AKT, the membranes were stripped and re-probed with antibodies to total AKT or actin.
Phospho akt ser473 4060
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-AKT Ser473 (4060) is a lab equipment product manufactured by Cell Signaling Technology. It is an antibody that specifically recognizes AKT protein phosphorylated at serine 473.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (2 mentions)
Akt 4691, by Cell Signaling Technology (2 mentions)
Phospho stat3 tyr705 9145, by Cell Signaling Technology (2 mentions)
Stat3 sc 482, by Santa Cruz Biotechnology (2 mentions)
Il 6 ab6672, by Abcam (2 mentions)
Odyssey fluorescent western blotting reagents, by Fortis Life Sciences (2 mentions)
Arid1a a301 041a, by Fortis Life Sciences (2 mentions)
Actin a 4700, by Merck Group (1 mentions)
Imagej software, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions)
Total akt 4691 s, by Cell Signaling Technology (1 mentions)
Total p44 42, by Cell Signaling Technology (1 mentions)
Total p38, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Ab97047, by Abcam (1 mentions)
Phospho mtor, by ZenBio (1 mentions)
Ab6728, by Abcam (1 mentions)
13 protocols using phospho akt ser473 4060
1
Immunoblotting for AKT and COX-IV
2
Western Blot Analysis of Protein Signaling
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Transfected PTC cells were lysed in RIPA buffer (Solarbio, China), and phenylmethylsulfonyl chloride was used as a protease inhibitor to stabilize the whole lysate. The extracted proteins were quantified by the bicinchoninic acid assay (Thermo Scientific, United States). Then the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BioRad, United States) followed by transferring them to the polyvinylidene difluoride (PVDF) membranes (Millipore, United States). The primary antibodies were as follows: MVP (16478-1-AP, Proteintech), phospho-AKTSer473 (4060S, Cell Signaling Technology), total-AKT (4691S, Cell Signaling Technology), phospho-mTOR (381557, Zen Bioscience), total-mTOR (380411, Zen Bioscience), Phospho-p44/42 (4370T, Cell Signaling Technology), total-p44/42 (4695T, Cell Signaling Technology), phospho-p38 (4511T, Cell Signaling Technology), total-p38 (8690T, Cell Signaling Technology), and -Actin (AP0060, Bioworld Technology). Primary antibodies were used for immunoblotting at 1:1,000 dilution. The membranes were then incubated with a secondary antibody (ab97047 or ab6728, Abcam). Eventually, proteins were detected by the chemiluminescence kit (Thermo Scientific), and images of the protein bands were quantified by ImageJ software (NIH, United States).
3
Antibody Immunoblotting for Insulin Signaling
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An antibody against IRS1 (sc‐8038, 1 : 1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho‐IRS1 (Ser612; 3193S, 1 : 1000), phospho‐AKT (Ser473; 4060S, 1 : 1000), AKT (4685S, 1 : 1000), β‐actin (4970 S, 1 : 1000), GAPDH (5174 S, 1 : 1000), and PPAR‐γ (2435 S, 1 : 1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). GPR50 (19762‐1‐AP, 1 : 1000) antibody was purchased from Proteintech (Rosemont, IL, USA).
4
Validation of Antibody Specificity
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The PKN1 antibody (ab195264) was purchased from Abcam (Cambridge, UK). FAK (#3285), phospho-FAK (Tyr397, #8556), Akt (#4685) and phospho-Akt (Ser473, #4060) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The beta-actin antibody (60008-1-lg) was purchased from Proteintech (Wuhan, China). All antibodies were validated by the company using western blots of human cell lines. The working dilution for each antibody is listed in Supplementary Table 8 . The western blot experiments were repeated independently for three times with similar results. The original full western blot plots are shown in Supplementary Fig. 9 .
5
Investigating ACTL6A and FSHR in AKT Signaling
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ACTL6A (ab131272) and FSH receptor (FSHR) (ab75200) antibodies were from Abcam (Cambridge, MA, USA). ACTL6A (sc-137062) mouse monoclonal antibody was from Santa Cruz (Dallas, TX, USA). AKT (#9272) and Phospho-AKT (Ser473) (#4060) antibodies were from Cell Signaling Technology (Danvers, MA, USA). PGK1 (17811-1-AP), GAPDH (10494-1-AP), and secondary antibodies were from Proteintech (Wuhan, China). FSH (869001) was from Merck (Darmstadt, Germany). LY294002 (S1105) and MK2206 (S1078) were from Selleck (Houston, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) kit (C0009) was obtained from Beyotime Biotech, Inc. (Shanghai, China). Transwell® Polycarbonate Membrane (#3422) was obtained from Corning (NY, USA).
6
Western Blotting Optimization Protocol
7
Characterization of anti-pUL32 Monoclonal Antibody
8
Western Blotting of Phosphorylated Akt
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Twenty micrograms of protein from each extract was loaded onto 8% to 12% SDS-polyacrylamide gel electrophoresis gels and transferred to PVDF membranes (iBlot, 2PVDF Regular Stacks, Invitrogen, Thermo Fisher Scientific, Inc.) Membranes were blocked in 5% w/v bovine serum albumin (BSA), 1X TBS and 0.1% Tween® 20 at room temperature for 1 h. The membrane was incubated with diluted primary antibody (phospho-Akt [Ser473] #4060, phospho-Akt [Thr308] [D25E6] #13038, or Akt #9272 HA-Tag [C29F4] #3724; 1:1000; Cell Signaling Technology) in TBS/Tween 20 (0.1% v/v) containing 5% BSA at 4 °C overnight. After washing, the membranes were incubated with a secondary antibody (anti-mouse IgG or anti-rabbit IgG; 1:2000; Thermo Fisher Scientific, Inc.) conjugated with horseradish peroxidase, followed by detection with enhanced chemiluminescence reagents (Bio-RAD Laboratories, Inc.).
9
Protein Extraction and Western Blot Analysis
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Tissue samples or cells were lysed in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a complete proteinase inhibitor (Roche Applied Sciences, Indianapolis, IN) and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Equal amounts of protein were resolved by SDS-PAGE and transferred to a PVDF membrane (BioRad, Hercules, CA) and blocked with anti-TGF-β1, ILK (Santa Cruz Biotechnology, Santa Cruz, CA), α-SMA (Sigma-Aldrich, St. Louis, MO), phospho-MAPK p44/42 (Thr202/Tyr204, #9101), total MAPK p44/42 (#9102), phospho-Akt (Ser473, #4060), and total Akt (#9272, Cell Signaling Technologies, Danvers, MA), followed by HRP–conjugated anti-rabbit or -mouse secondary antibodies (#7074 or #7076, Cell Signaling Technology, Danvers, MA). The blots were developed using an ECL system (PerkinElmer, Boston, MA). GAPDH (#2118, Cell Signaling Technology, Danvers, MA) was used as the loading control. The films were scanned and quantitative analysis of the ratio of phosphorylated to total MAPK p44/42, Akt or TGF-β1, ILK, and α-SMA from three independent experiments using Kodak 1D 3.5 software (Rochester, NY).
10
Astragaloside IV Modulates Immune Response
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Astragaloside IV (purity > 98%) was purchased from Ronghe Co. (Shanghai, China). OVA (purity > 98%, Grade V) and methacholine (purity > 98%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Imject alum adjuvant was obtained from Thermo Fisher Scientific Co. (Waltham, MA, USA). Dexamethasone sodium phosphate injection (purity > 99%; 5 mg/mL) was provided by Tianjin pharmaceutical group Xinzheng Co. (Tianjin, China). Rapamycin (purity > 98%) was purchased from LC Laboratories (Woburn, MA, USA). ELISA kits for INF-γ, IL-4, IL-5, IL-13, and IL-17A, FITC-labeled anti-rat CD4, APC-labeled anti-rat CD25, and PE-labeled anti-rat Foxp3 were purchased from eBioscience Co. (San Diego, USA). Antibodies for phospho-S6 ribosomal protein (Ser235/236) #4858, phospho-Akt (Ser473) #4060, Akt (#4691), phospho-4E-BP1 (Ser65) #9451, phospho-p70 S6 Kinase (Thr389) #9234, and β-actin were obtained from Cell Signaling (Danvers, MA, USA).
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