Animal experiments were in compliance with the ethical regulations set by UC Berkeley Animal Care and Use Committee. Male C57BL/6 (wild type) mice (Jackson) or B6 Cg-Lepob (ob/ob) (Jackson) were used at 8 wks of age. Neither randomization nor blinding was employed. A sample size of 5 per group was initially used to assess whether differences could be detected and was found to be sufficient to achieve statistical significance. For fasting and refeeding experiments, mice were fasted overnight and then fed a high carbohydrate, fat free diet for 6 hrs. For knockdown or overexpression experiments, mice received through tail vein injection 100μl of adenovirus (MED17 shRNA, MED17 WT, MED17 S53A, or CK2α1 shRNA; Vector Biolabs) at 2.0 × 1010 PFU/mL. Twelve days after injection mice were fasted overnight then refed high carbohydrate diet for 6 hrs. Knockdown or overexpression was verified by measuring mRNA or protein abundance prior to qPCR analysis of lipogenic markers. For CK2 inhibition experiments, mice were injected with either vehicle or CX-4945 (75mg/kg; APEX Bio) intraperitoneally for 5 days, then fasted overnight and refed high carbohydrate diet for 6 hrs.
Cx 4945
Manufactured by Apexbio
Sourced in United States
CX-4945 is a selective and potent inhibitor of the serine/threonine protein kinase CK2 (Casein Kinase 2). It functions by binding to the ATP-binding site of the CK2 enzyme, thereby inhibiting its catalytic activity.
Automatically generated - may contain errors
Lab products found in correlation
B6 cg lepob ob ob, by Jackson ImmunoResearch (1 mentions)
Wild type c57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Hct116, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Forskolin, by Fujifilm (1 mentions)
Sp600125, by Apexbio (1 mentions)
Trametinib, by Apexbio (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Silmitasertib, by Apexbio (1 mentions)
6 protocols using cx 4945
1
Fasting and Refeeding Regulation of Lipogenesis
2
Modulation of Microglia-Like Cells
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MGLs generated from WT/WT and ΔE9/ΔE9 hiPSCs were plated into a 48-well plate coated with poly-L-lysine (# P6282; Sigma) at a cell density of 200,000 cells/well. After 24 h, MGLs were treated with either DMSO (#D8418; Sigma) or lipopolysaccharides (LPS, 100 ng/ml) (#4391; Sigma) with or without a casein kinase inhibitor, CX-4945 (#A8330; ApexBio Technology; 0.5 μM) or SGC-CK2-1 (Axtman lab; 10 nM), and cultured for 24 h. At the end of 24 h, RNA, protein, and condidtioned medium were harvested from the MGLs.
3
Cell Culture and Compound Treatments
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Cell lines were purchased from American Type Culture Collection. The 293T, MCF7, and HCT116 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37°C with 5% CO2. SEM was maintained in IMDM with 10% FBS. MV4-11 and RS4;11 were maintained in RPMI 1640 supplemented with 10% FBS and nonessential amino acids. For the CHX chase experiment, indicated concentrations of CHX (Sigma) or MG132 (Sigma) were added to the culture medium at the indicated time points before harvesting the cells. For CKII inhibitor treatment, the indicated concentrations were added to the cell culture for 30 h before harvesting the cells for a Western blot or an in vitro taspase1 assay. CX-4945, TTP22, DMAT, and TTB were purchased from ApexBio.
4
Cell Treatment with Signaling Modulators
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5
Pharmacological Inhibition of Signaling Pathways
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For in vitro and in vivo studies, JNK inhibitor SP600125, MEK inhibitor Trametinib (GSK1120212), MLK inhibitor URMC-099, and CK2 inhibitor CX-4945 (Silmitasertib) were purchased from APExBIO (A4604, A3018, B4877, A833010, respectively). p38 inhibitor SB203580 was purchased from Selleckchem (Cat No S1076) for the in vitro experiments. For in vivo studies, water soluble SB203580 hydrochloride was purchased from APExBIO (B1285). SW-538 (SW034538) was provided by Elizabeth J. Goldsmith et al.
6
Protein Stability Assay Protocol
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Protein stability assays were performed as described elsewhere [23 (link),24 (link)]. Briefly, cells (1 × 106 cells/well) were seeded into 12-well plates, cultured overnight at 37 °C and 5% CO2 in DMEM-F12 medium supplemented with 10% FBS, and incubated overnight with 20 μg/mL cycloheximide (CHX, Tocris, Bristol, Avon, UK) to inhibit protein synthesis. Then, cells were incubated in the presence of 25 μM silmitasertib (a selective CK2 inhibitor, formerly known as CX-4945; Apexbio, Houston, TX, USA) or vehicle only (0.001% DMSO) for 6 h. Cells were then harvested and lysed, and the total protein was analyzed using Western blots.
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