Western blotting was performed as described previously with minor modifications.34 In brief, HUVEC or exosomal proteins were extracted and incubated overnight at 4°C with primary antibodies including anti‐CD9 (1:1000; Cell Signaling Technology, Danvers, MA, USA; #13174), anti‐CD81 (1:1000; Abcam, Cambridge, Britain; ab109201), anti‐ALIX (1:1000; Cell Signaling Technology; #2171), anti‐VEGF (1:1000; Abcam; ab46154), anti‐Ang‐1 (1:500; Abcam; ab8451), anti‐PIK3R2 (1:1000; Abcam; ab131067), anti‐phosphorylated Akt (p‐Akt; 1:1000, Cell Signaling Technology; #4060), anti‐Akt (1:1000, Cell Signaling Technology; #4685) and anti‐β‐actin (1:2000; Proteintech, Rosemont, USA; #60008‐1‐lg). After incubation with horseradish peroxidase‐conjugated secondary antibodies (1:5000, ProteinTech; SA00001‐1 or SA00001‐2) at room temperature for 2 hours, the blots were visualized using chemiluminescence substrate (ECL kit; Beyotime, Shanghai, China) and the quantification of protein bands was analysed with ImageJ software. β‐actin was used as the loading control for internal normalization.
Anti phosphorylated akt p akt
Manufactured by Cell Signaling Technology
Sourced in United States
Anti‐phosphorylated Akt (p‐Akt) is a primary antibody that specifically recognizes the phosphorylated form of the Akt protein. Akt is a serine/threonine protein kinase that plays a central role in the regulation of cellular processes such as metabolism, growth, proliferation, and survival.
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Lab products found in correlation
Anti akt, by Cell Signaling Technology (5 mentions)
Anti pi3k, by Cell Signaling Technology (2 mentions)
Anti pten, by Cell Signaling Technology (2 mentions)
Ecl kit, by Beyotime (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti alix, by Cell Signaling Technology (1 mentions)
Anti vegf, by Abcam (1 mentions)
Anti cd9, by Cell Signaling Technology (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Anti cd81, by Abcam (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Anti s6k1, by Cell Signaling Technology (1 mentions)
Anti mtorc1, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit antibody, by Jackson ImmunoResearch (1 mentions)
Anti cd68, by BD (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti nrf2, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Wuhan Servicebio Technology (1 mentions)
Anti cleaved caspase3 c caspase3, by Cell Signaling Technology (1 mentions)
8 protocols using anti phosphorylated akt p akt
1
Western Blotting Analysis of HUVEC Exosomal Proteins
2
Protein Isolation and Western Blot Analysis
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Protein isolation and Western blotting have been described before [23 (link)]. The primary antibodies for Western Blot were anti-CD68 (1:800; Becton-Dickinson Biosciences), anti-LC3 (1:750; Cell Signaling, San Jose, CA, USA), anti-PI3K (1:750; Cell Signaling), anti-phosphorylated PI3K (pPI3K, 1:1000; Cell Signaling), anti-AKT (1:1000; Cell Signaling), anti-phosphorylated AKT (pAKT, 1:1000; Cell Signaling), anti-mTORC1 (1:1000; Cell Signaling), anti-S6K1 (1:500; Cell Signaling), anti-phosphorylated S6K1 (pS6K1, 1:1000; Cell Signaling) and anti-α-tubulin (1:1000; Cell Signaling). The secondary antibodies were HRP-conjugated anti-rabbit antibodies (Jackson ImmunoResearch Labs, West Grove, PA, USA). Western blot quantification was performed using NIH ImageJ software (Bethesda, MA, USA) using 4-5 repeats.
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cells via radioimmunoprecipitation assay buffer (Beyotime Biotechnology) after different treatments, then equally separated by 10% SDS-PAGE gel, and ultimately transferred into nitrocellulose membranes (GE Healthcare Life Sciences, Beijing, China). Membranes were blocked in 5% nonfat milk for 1 h, and incubated overnight at 4°C with anti-phosphorylated-AKT (P-AKT, 1:1,000, Cell Signaling Technology), anti-AKT (1:1,000, Cell Signaling Technology), anti-P-GSK3β (1:1,000, Cell Signaling Technology), anti-GSK3β (1:1,000, Cell Signaling Technology), anti-FYN (1:1,000), anti-NRF2 (1:1,000), anti-BAX (1:1,000, Cell Signaling Technology), anti-BCL-2 (1:1,000, Abcam), anti-CLEAVED CASPASE-3 (C-CASPASE 3, 1:1,000, Cell Signaling Technology), anti-GAPDH (1:1,000, Servicebio technology), and anti-β-ACTIN (1:2000, Servicebio technology). The catalogs of antibodies are shown in Supplementary Table S1 .
4
Western Blot Analysis of Signaling Pathways
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Protein was extracted from the cultured cells with RIPA lysis buffer (1% NP40, 0.1% Sodium dodecyl sulfate (SDS), 100 μg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, in PBS) on ice. Protein concentration was determined using a BCA protein assay kit (Bio-rad, China), and whole lysates mixed with 4×SDS loading buffer (Bio-rad, China) were denatured by 5 minutes’ incubation at 100°C for 5 min. Afterwards, proteins were separated on SDS-polyacrylamide gels, and then transferred to a PVDF membrane. The membrane blots were first probed with a primary antibody. After incubation with horseradish peroxidase-conjugated second antibody, protein was visualized using an enhanced chemiluminescent system. Primary antibodies were rabbit anti-phosphorylated p38 (p-p38), anti-p38, anti-phosphorylated ERK1/2 (p-ERK1/2), anti-ERK1/2, anti-phosphorylated AKT (p-AKT) and anti-AKT (all purchased from Cell Signaling, San Jose, CA, USA). Secondary antibody is HRP-conjugated anti-rabbit (Jackson ImmunoResearch Labs, West Grove, PA, USA).
5
Western Blot Analysis of EV Proteins
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Equivalent amounts of protein from whole-cell lysates or EVs were loaded into each lane of SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes (Hybond-P; GE Health Care, Buckinghamshire, UK). Membranes were incubated in Blocking One or Blocking One-P (Nacalai Tesque, Inc., Kyoto, Japan) for 30 min before being incubated in the following primary antibodies overnight at 4 °C: anti-CD9 (Chemicon International Inc., Temecula, CA, USA), anti-CD63 (BD Biosciences), anti-CD81 (BioLegend), anti-signal transducer and activator of transcription 3 (STAT3; Cell Signaling Technology), anti-phosphorylated STAT3 (p-STAT3; Cell Signaling Technology), anti-Akt (Akt; Cell Signaling Technology), anti-phosphorylated Akt (p-Akt; Cell Signaling Technology), and anti-β-actin antibody (Sigma-Aldrich). The membranes were subsequently incubated with secondary antibodies for 60 min at room temperature. Peroxidase activity of secondary antibodies was detected using ECL prime Western Blotting Detection Reagent (GE Healthcare UK Ltd.) and visualized using an Amersham Imager 600 (GE Healthcare UK Ltd.) according to the manufacturer’s protocol.
6
Mouse Model of Glaucoma Evaluation
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Thirty-five, 8- to 10 week-old female C57BL/6 mice (18 to 20 g, purchased from Hyochang Science, Daegu, Korea) were used for this study. These mice were kept in the facility with a standard environment throughout the study as follows: room temperature 25 ± 1℃, relative humidity 60 ± 10%, and alternating 12-hour light/dark cycles (8 a.m. to 8 p.m.). All experimental procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Based on the clinical evaluations (described below), the mice were then randomly divided into seven groups (5 mice for each group): normal saline-treated control group, BAC-treated group and five anti-glaucomatic agent-treated groups.
Anti-TNF-α, anti-IL-6 and anti-HLA DR were purchased from Abcam (Cambridge, MA, USA). Anti-phosphorylated c-Jun NH(2)-terminal kinase (pJNK) and anti-phosphorylated-Akt (pAkt) were purchased from Cell Signaling Technology (Allschwil, Switzerland). 4',6-diamidino-2-phenylindole (DAPI) and Prolong Gold were purchased from Invitrogen (Carlsbad, CA, USA).
Anti-TNF-α, anti-IL-6 and anti-HLA DR were purchased from Abcam (Cambridge, MA, USA). Anti-phosphorylated c-Jun NH(2)-terminal kinase (pJNK) and anti-phosphorylated-Akt (pAkt) were purchased from Cell Signaling Technology (Allschwil, Switzerland). 4',6-diamidino-2-phenylindole (DAPI) and Prolong Gold were purchased from Invitrogen (Carlsbad, CA, USA).
7
Western Blot Analysis of Signaling Proteins
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Cell lysates were extracted using radioimmunoprecipitation assays (Beyotime, Shanghai, China), fractionated by electrophoresis, and transferred onto a polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% non-fat skim milk and incubated with primary and secondary antibodies. The bands on the membranes were visualized using electrochemiluminescence (Pierce, Rockford, IL, USA). The following primary antibodies were used: anti-KMT2D (1:1,000; Abcam, Cambridge, MA, USA), anti-phosphatase and tensin homolog (PTEN; 1:1,000; Cell Signaling Technology, Danvers, CO, USA), anti-PTEN (1:1,000), anti-phosphoinositide 3-kinases (PI3K) (1:1,000; Cell Signaling Technology), anti-protein kinase B (AKT; 1:1,000; Cell Signaling Technology), and anti-phosphorylated AKT (pAKT; 1:1,000; Cell Signaling Technology).
8
Western Blot Analysis of PTEN-PI3K-AKT Pathway
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Cells lysates extracted using radioimmunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) were fractionated by electrophoresis and transferred on a polyvinylidene uoride (PVDF) membrane (Millipore).
The membrane was blocked in 5% nonfat skim milk and incubated with primary antibodies and secondary antibodies. Bands on the membrane were visualized by electrochemiluminescence (ECL; Pierce, Rockford, IL, USA). The primary antibodies used were as follows: anti-PTEN (1:1000), anti-PTEN (1:1000), anti-PI3K (1:1000), anti-AKT (1:1000), anti-phosphorylated-AKT (pAKT; 1:1000), (Cell Signaling Technology, Danvers, CO, USA).
The membrane was blocked in 5% nonfat skim milk and incubated with primary antibodies and secondary antibodies. Bands on the membrane were visualized by electrochemiluminescence (ECL; Pierce, Rockford, IL, USA). The primary antibodies used were as follows: anti-PTEN (1:1000), anti-PTEN (1:1000), anti-PI3K (1:1000), anti-AKT (1:1000), anti-phosphorylated-AKT (pAKT; 1:1000), (Cell Signaling Technology, Danvers, CO, USA).
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