Image studio ver 4

At designated time points after cytokine stimulation, total cell lysates from BMDMs or BMDCs were washed three times with room temperature PBS. For experiments including cytokine stimulation followed by rest, BMDMs or BMDCs were treated for 30 min with designated concentration of cytokine, washed once with room temperature PBS, and cultured in cytokine-free media for specified time of rest. The cells were lysed in the culture dish using 0.02% NP-40 supplemented with HALT protease and phosphatase inhibitors (Thermo Fisher Scientific) and 1× SDS–PAGE buffer (0.0625 M Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-ME, and 0.01% bromophenol blue) was added. Equivalent protein amounts were loaded into 10% acrylamide gels and transferred onto Polyvinylidene difluoride membranes (Millipore). Blots were probed for IFNGR1 (K17), pSTAT1Y⁷⁰¹ (58D6; Cell Signaling), or Total STAT1 (91-C; Cell Signaling) with β-Actin (8H10D10; Cell signaling) as a loading control on each blot. Blots were developed using the secondary antibodies goat α-rabbit IR 800 (926–32211; LI-COR) and goat α-mouse IR 680 (926–68070; LI-COR) and imaged on an Odyssey CLX (LI-COR). All pSTAT1Y⁷⁰¹ and IFNGR1 bands were normalized to β-Actin on the same blot using ImageStudio ver 4.0 software (LI-COR). Densitometry graphs are pooled from at least three independent pSTAT1Y⁷⁰¹ or IFNGR1 blots.

Naïve peritoneal macrophages were treated with indicated concentrations of IFNγ or IFNβ for either 0, 5, 30, 60 minutes. Cells were lysed in 0.02% NP-40 supplemented with HALT protease and phosphatase inhibitors (Thermo Scientific) directly in the tissue culture dish. Protein concentrations were determined by BCA protein assay (Pierce) and 1x SDS-PAGE buffer (0.0625 M Tris-Cl, pH 6.8/2% SDS/10% glycerol/5% 2-ME/0.01% Bromophenol Blue) was added. Equivalent protein amounts were loaded into 10% acrylamide gels and transferred onto PDVF membranes (Millipore). Blots were probed for pY701 STAT1 (58D6, Cell Signaling) or Total STAT1 (91-C, Cell Signaling) with β-Actin (8H10D10, Cell signaling) as a loading control on each blot. Blots were developed using the secondary antibodies, goat α-rabbit IR 800 (926–32211, LI-COR) and goat α-mouse IR 680 (926–68070, LI-COR), and imaged on an Odyssey CLX (LI-COR). All pSTAT1 bands were normalized to β-Actin on the same blot using ImageStudio Ver 4.0 software (LI-COR). Densitometry graphs are pooled from at least 3 independent pSTAT1 blots.