Mog (HPLC > 98%) was obtained from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China; CAS:88901-36-4). The compound was dissolved in dimethyl sulfoxide (DMSO, 0.1% final concentration in cultural medium). DMSO, LPS (Escherichia coli Serotype 055:B5), and LY294002 (10 μM, AKT inhibitor) were purchased from Sigma Chemical Co. (St Louis, USA). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and phosphate-buffered saline (PBS) were supplied by Coolaber (Beijing, China). BCA were obtained from Beyotime Biotechnology (Shanghai, China). ECL chemiluminescence substrates were supplied from Millipore Corporation (Billerica, USA). COX-2, iNOS, TLR4, Nrf2 HMGB1, IL-1β, IL-18, NF-κB, p-NF-κB, MyD88, p-AKT, AKT, p-AMPK, AMPK, IL-6, and TNF-α were obtained from ABclonal Technology (Wuhan, China). Rabbit polyclonal antibodies against ERK (#4695), p-ERK (#4370), JNK (#9252), p-JNK (#4668), p38 MAPK (#8690), p-p38 (#4511), IκBα (#4814), and p-IκBα (#2859) were purchased from Cell Signaling Technology (Bossdun, USA). NQO1 (ab80588), HO-1 (ab68477), GCLC (ab207777), and GCLM (ab126704) were provided by ABCAm (Cambridge, United Kingdom). Dulbecco's modified Eagle's medium (DMEM) and trypsin were provided by Gibco (USA). Fetal bovine serum (FBS) was from Biological Industries (Uruguay, South America).
P ampk
Manufactured by ABclonal
Sourced in China
P-AMPK is a recombinant protein that corresponds to the phosphorylated form of the AMP-activated protein kinase (AMPK). AMPK is a critical cellular energy sensor that plays a key role in regulating cellular metabolism. P-AMPK represents the activated state of AMPK, which is induced by phosphorylation.
Automatically generated - may contain errors
Lab products found in correlation
Cox 2, by ABclonal (2 mentions)
Gapdh, by ABclonal (2 mentions)
β actin, by ABclonal (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Ly294002, by Merck Group (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Phosphate buffered saline (pbs), by Coolaber (1 mentions)
P nf κb, by ABclonal (1 mentions)
P akt, by ABclonal (1 mentions)
Tnf α, by ABclonal (1 mentions)
Il 1β, by ABclonal (1 mentions)
Il 18, by ABclonal (1 mentions)
Nf κb, by ABclonal (1 mentions)
Myd88, by ABclonal (1 mentions)
Interleukin 6 (il 6), by ABclonal (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
7 protocols using p ampk
1
Mog Modulates Inflammatory Pathways
2
Western Blot Analysis of Mitochondrial Proteins
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The cerebral ischemic cortex was homogenized in RIPA buffer. The homogenized protein was centrifuged at high speed, then the supernatant was extracted and the precipitation was discarded. Then the total protein concentration was determined with the BCA kit. 20 μg total protein samples from cortex were separated using SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked using blocking solution (5% non-fat milk in TBST, pH 7.4) at room temperature for 60 min. Discard blocking solution and incubation with the primary antibody overnight at 4°C. Primary antibodies included: Opa1 (1:2000, Proteintech), Mfn1 (1: 2000, Proteintech), Mfn2 (1: 2000, Proteintech) and β-actin (1: 5000, Proteintech), AMPK (1:4000, Abclonal), pAMPK (1:5000, Abclonal), NDUFB8 (1:500, Abclonal), SDHB (1:500, Abclonal), UQCRC2 (1:500, Abclonal), MTCO2 (1:500, Abclonal), ATP5A1 (1:500, Abclonal). The following day, membranes were washed with TBST (10 min × 3) each time and subsequently incubated with secondary antibodies (1: 8000) for 2 h at room temperature. Membranes were then washed three times with TBST (10 min × 3). Protein expression was examined by using Chemiluminescent HRP Substrate (Millipore) and Cytiva. Densitometric values was quantified with ImageJ and were normalized with respect to β-actin immunoreactivity to correct for any loading and transfer differences among samples.
3
6-OHDA-induced Neuroinflammation Model
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VPA was obtained from Sigma-Aldrich (Saint Louis, MO, United States of America (USA)). 6-OHDA, L-ascorbic acid and LPS were obtained from (Medchemeexpress, USA). Ketamine and xylazine was obtained from Prince Naif bin Abdul-Aziz Health Research Center, King Saud University (KSU), Saudi Arabia (SA). Optimal Cutting Temperature (OCT) was obtained from (ThermoFisher Scientific, USA). Trizol reagent was obtained from (Invitrogen, USA). SYBR Green, PCR Master Mix, and cDNA reverse transcription kit were bought from (Medchemeexpress, USA). IL-6, TNF-α, and NF-κB primers were created and customized by (integrated DNA Technologies (IDT), Belgium). Cocktail protease inhibitors and RIPA lysis buffer were purchased from (ThermoFisher Scientific, USA). Bicinchoninic acid assay (BCA assay) was purchased from (Molequle-on, Auckland, New Zealand). Primary rabbit polyclonal antibodies COX-2, GPx-1, p-AMPK, AMPK, and GAPDH and secondary horseradish peroxidase goat antirabbit IgG antibodies were purchased from (ABclonal Technology, USA).
4
Western Blot Analysis of Protein Expression
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Cells were collected and lysed with a lysis buffer containing protease inhibitors and phosphatase inhibitors to extract total protein. The target protein separated by SDS-polyacrylamide gel, and transferred to NC membrane. Then the Amersham Imager 600 (GE Healthcare, Little Chalfont, UK) were used to detect specific protein bands. Primary antibodies included antibodies against β-Actin (ABclonal, AC026, 1:200,000), TRPV4 (Proteintech, 20987-1-AP; 1:2000), P-ERM (abcam, ab76247; 1:1000), P-MLC (Cell Signaling Technology, 3671 T; 1:1000), P-AMPK (ABclonal, AP1002; 1:2000), ERM (abbkine, ABP55285; 1:2000), MLC (Cell Signaling Technology, 3672; 1:2000), AMPK (ABclonal, A1229; 1:2000), ROCK1 (Cell Signaling Technology, 4035 T; 1:1000), E-Cadherin (Cell Signaling Technology, 3195 T; 1:1000), N-Cadherin (Cell Signaling Technology, 13116 T; 1:1000), Vimentin (Cell Signaling Technology, 5741 T; 1:1000), MMP2 (ABclonal, A19080, 1:800), MMP9 (ABclonal, A0289, 1:600). Western blotting assay for each protein was performed at least three times.
All the full and uncropped western blots was shown in Supplemental Material of Original Western Blots Data.
All the full and uncropped western blots was shown in Supplemental Material of Original Western Blots Data.
5
Protein Expression Analysis in MDA-MB-231 Cells
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MDA-MB-231 cells were cultured or administrated for 36 h and then lyzed using lysis buffer and the total protein samples were isolated and eluted with SDS buffer, separated by SDS-polyacrylamide gels, and electroblotted onto PVDF membranes. The specific protein bands were detected with Odyssey Scanning System (LI-COE Inc., Superior St., Lincoln, NE) according to the previous report [27] (link). The following are the origins of the primary antibodies and the corresponding diluting proportions. (1) G6PD from Abclonal Technology, 1:2000, was used to determine the level of PPP; (2) β-actin from Santa Cruz Biotechnology, 1:2000, was used as internal reference; (3) p-ACC from Cell Signaling Technology, 1:1000, was used to determine the phosphorylation of ACC and the activation of AMPK; (4) CPT1A from Abclonal Technology, 1:2000, was used to determine the level of FAO; (5) PDH from Cell Signaling Technology, 1:1000, was used to determine the level of acetyl-CoA produced from glucose; (6) LDH from Abclonal Technology, 1:2000, was used to determine the level of glycolysis; (7) AMPK from Abclonal Technology, 1:2000, was used to determine the expression level of AMPK and (8) p-AMPK from Abclonal Technology, 1:2000, was used to determine the level of AMPK activation. Western blot assays for each protein were performed at least three times
6
Autophagy Regulation in Neurodegeneration
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EGCG, anti-LC3B antibody, and anti-NeuN antibody were purchased from Sigma-Aldrich (USA). Donkey anti-mouse IgG secondary antibody was obtained from Life Technologies (Thermo Fisher Scientific, United States). Phosphor-AKT (p-AKT), AKT, p-AMPK, AMPK, p-mTOR, mTOR, beclin1, β-actin, and GAPDH were obtained from ABclonal Technology Co., Ltd. (China). P62/SQSTM1 polyclonal antibody was obtained from Proteintech (China). Horseradish peroxidase (HRP)-conjugated secondary antibody was obtained from Cell Signaling Technology (United States). GSK690693, rapamycin, and LY294002 were obtained from MedChemExpress (United States). Fetal bovine serum (FBS) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Gibco (Thermo Fisher Scientific, United States). The antifade mounting medium was obtained from Solarbio (China). RIPA lysis buffer was obtained from Beyotime (China). Protease and phosphatase inhibitor cocktails and NcmBlot blocking buffer were obtained from New Cell and Molecular Biotech (China). BCA protein assay kit was obtained from CWBIO (China). Cell Counting Kit-8 (CCK8) and ECL chemiluminescent HRP substrate A&B were obtained from Antgene (China). All other reagents, unless stated otherwise, were obtained from Biosharp (China).
7
Immunoblotting Analysis of Cellular Proteins
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Radioimmunoprecipitation assay buffer (RIPA) buffer purchased from Beyotime Biotechnology (China) was used to extract total protein from tissues and cultured cells, and the supernatant was collected after centrifugation (12,000×g, 10 min, 4°C). After blocking with 5% skim milk, the membrane was incubated with primary antibodies against LETM1 (1:500, Santa Cruz, USA), AMPK (1:500, ABclonal, China), p-AMPK (1:500, ABclonal, China), Bcl-2 (1:500, ABclonal, China), p-Bcl-2 (1:500, Proteintech, USA), Bax (1:500, ABclonal, China), caspase-3 (1:500, Proteintech, USA), Beclin-1 (1:500, Proteintech, USA), p62 (1:500, Proteintech, China), LC3 (1:500, Proteintech, USA), and β-actin (1:2000, ABclonal, China) overnight. The membrane was then incubated with secondary antibodies (1:2,000, Proteintech, China) for 2 h the next day. The protein bands on the blot were analyzed.
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