PHD2 (Novus Biologicals), VEGFA (Proteintech), HIF1α, HIF2α, Lamin β1 (Abcam), GAPDH, β-actin (Santa Cruz), mTOR, pmTOR, Akt, pAkt and pS70S6K (Cell Signaling technologies) were assessed 24h post IL-4 stimulation (10 ng/ml) in scramble siRNA and Mt3 siRNA treated Mϕ and/or WT and Mt3−/− Mϕ. For total STAT6 and pSTAT6 (BD Biosciences) analysis, WT and Mt3−/− Mϕ were stimulated with IL-4 for 15 min; for total STAT1 and pSTAT1 (Abcam) analysis, Mϕ were stimulated with 10 ng/ml IL-4 for 24h, followed by change of media and 5 ng/ml IFNγ for 15 min. To determine the translocation of HIF, total cytosolic and nuclear proteins were isolated using NE-PER® Nuclear and Cytoplasmic Extraction Reagents according to manufacturer’s instructions (Thermo Scientific). Cell lysates were prepared using Denaturing Cell Extraction Buffer (Invitrogen). Total cell lysates as well as cytosolic and nuclear proteins were run on 4%–20% Precise Protein Gels (Pierce) and transferred on to nitrocellulose membranes. Western blots were probed with corresponding host specific HRP conjugated secondary antibodies and developed using SuperSignal West Femto Chemiluminescent Substrate (Thermofisher).
Lamin β1
Manufactured by Abcam
Lamin β1 is a structural protein that is a component of the nuclear lamina, a fibrous layer on the inner side of the nuclear envelope of eukaryotic cells. It provides mechanical support and organization to the nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Vegfa, by Proteintech (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Immunobilon p, by Merck Group (1 mentions)
Nrf2 primary antibody, by Proteintech (1 mentions)
α tubulin, by GeneTex (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
2 protocols using lamin β1
1
Macrophage Polarization Signaling Pathways
2
Nrf2 Expression Analysis in HepG2 Cells
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Nuclear and cytoplasmic protein extracts were harvested from HepG2 cells treated with or without PME (100 μg/mL) for 6 h and were prepared as described [12 ]. Protein samples (20 μg) were separated using electrophoresis on a 4%–20% gradient gel prepared by a TOOLS HR Gradient gel solution (TOOLS Biotech, New Taipei City, Taiwan) and transferred to a polyvinylidene difluoride transfer membrane (Immunobilon-P, Millipore, Bedford, MA). Subsequently, the membrane was blocked using 2% bovine serum albumin and then incubated with an Nrf2 primary antibody (1:1000, ProteinTech, Chicago, IL) or lamin β1 (1:1000, Abcam, Cambridge, MA) and α-tubulin (1:1000 GeneTex, Irvine, CA) at 4°C overnight. After incubating with a secondary antibody, the Immobilon™ Western Chemiluminescent HRP Substrate (Merck-Millipore) was used to detect reactive protein signals.
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