Lysates were prepared in RIPA buffer (Cell Signaling Technologies) with 1mM PMSF per manufacturer's instructions. Proteins were separated on a NuPAGE gel (Invitrogen) and transferred to a PVDF membrane using the XCell II blotting system (Invitrogen). Membranes were blocked with 5% nonfat milk and incubated with primary antibody overnight at 4°C. Primary antibodies were purchased from Cell Signaling Technologies: IRAK1 (#4504), phospho-IkBa (#2859), IkBa (#4814), phospho-p38 MAPK (#4511), p38 MAPK (#8690), phospho-p44/42 MAPK (ERK1/2, #4370), p44/p42 MAPK (ERK1/2, #4695), NFkB2 p100/p52 (#4882), phospho-NFkB p65 (#3033), NFkB p65 (#8242) phospho-TBK1 (#5483), TBK1 (#3504). CXCL1 (PA1-29220, Thermo Fisher Scientific), GAPDH (CB1001), and total actin antibody (MAB1501) were purchased from EMD Millipore. HuR (sc-5261), IRAK1 (sc-5288), phospho-MAPKAPK2 (sc-293139), MAPKAPK2 (sc-393609), and TTP (sc-374305) were purchased from Santa Cruz Biotechnology. TRAF2 (592) was purchased from Medical & Biological Laboratories. Following washing, membranes were incubated with HRP-tagged anti-mouse IgG (1706516, Bio-Rad) or anti-rabbit IgG (NA934, GE Healthcare) and developed using SuperSignal West Pico or Femto substrate (Thermo Fisher Scientific).
Phospho ikba
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-IkBa is a laboratory reagent that detects the phosphorylated form of the IκBα protein. IκBα is a regulatory protein that inhibits the NF-κB transcription factor. Phosphorylation of IκBα leads to its degradation, allowing NF-κB to translocate to the nucleus and regulate gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Phospho nf kb p65, by Cell Signaling Technology (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Nf kb p65, by Cell Signaling Technology (1 mentions)
P44 p42 mapk, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Nf kb2 p100 p52, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk, by Cell Signaling Technology (1 mentions)
Gapdh cb1001, by Merck Group (1 mentions)
Irak1, by Cell Signaling Technology (1 mentions)
Phospho tbk1, by Cell Signaling Technology (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Gel pro analyzer 6, by Media Cybernetics (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Total ikba, by Cell Signaling Technology (1 mentions)
Supersignal west pico substrate, by Thermo Fisher Scientific (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
3 protocols using phospho ikba
1
Immunoblotting Protocol for Inflammatory Signaling
2
Extracellular DNA Activation of BMDM
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BMDMs generated as aforementioned were seeded onto 6-well plates at a density of 2 × 105 cells/well and serum starved overnight. Cell-free DNA was secreted by AE17, or AE17 cells treated with karonudib (10 μM, overnight) isolated from culture supernatants using a commercial kit (Nucleospin, Macherey-Nagel Düren, Germany). BMDMs were subsequently treated with 20 ng/mL extracellular DNA for 2 h. TLR9 inhibitor chloroquine (Merck, Darmstadt, Germany) was used at 2 μg/mL for 40 min before addition of cfDNAs. Cell lysates were prepared and analyzed by Western blotting for phospho-p65 NFKB (#3031), total p65 NFKB (#4764), phospho-ikBa (#2859) and total-ikBa (#4812) (Cell Signaling Technology Inc., Danvers, MA, USA). Results were normalized to actin (#4970, Cell Signaling Technology Inc., Danvers, MA, USA). Visualized bands were quantified by GelPro Analyzer 6.3 (Media Cybernetics, L.P., Rockville, MD, USA)
3
Western Blot Analysis of Signaling Pathways
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Western blot analyses on liver samples were performed as previously described (8 ) for the NF-κB and protein kinase B/mechanistic target of rapamycin (AKT/MTOR) signaling pathways [primary antibodies detecting respectively NF-κB p65, phospho-NF-κB p65 (Ser536), NF-κB inhibitor α (IKBA) and phospho-IKBA (Ser32), AKT, phospho-AKT (Ser473), MTOR and phospho-MTOR (Ser2448); Cell Signaling Technology). beta-actin was used as control protein (Abcam). Signals were revealed using the SuperSignal West Pico substrate with Femto Chemiluminescent substrate addition for the NF-κB pathway (Thermo Fisher Scientific), and analyzed with the ImageQuant TL instrument and software v.8.1 (GE Healthcare).
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