Tripartite transcription factor cooperativity anchors mSWI/SNF to poise the T cell effector landscape prior to antigen receptor expression. Gamble, Bradu, Caldwell et al. (2023)
Original Western Blot images from indicated figures in Gamble, Bradu, Caldwell et al. (2023) "Tripartite transcription factor cooperativity anchors mSWI/SNF to poise the T cell effector landscape prior to antigen receptor expression." File names indicate figure panel and target proteins.
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Preparation of nuclear extracts: CCRF-CEM cells or thymocytes from 3-6 week-old mice were liberated as above and resuspended in Thymocyte Hypotonic Buffer supplemented with 1 mM DTT, 1 mM PMSF, and 1x cOmplete protease inhibitor cocktail (Roche). Following 15-minute hypotonic lysis on ice, nuclei were centrifuged at 700 RCF and resuspended in 600uL per 200e6 cells of Buffer C (10 mM HEPES pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 3 mM MgCl2, 10% Glycerol). Ammonium sulfate was added drop-wise to 150 mM and samples rotated at 4°C for 30 minutes. Lysates were centrifuged at 435,400 RCF (100,000 RPM) in TLA 120.2 rotor (Beckman Coulter) for 10 minutes at 4°C. Supernatant was transferred to an ice-chilled tube and proteins were precipitated with 0.33 mg/L ammonium sulfate on ice for 20 minutes. Lysates were centrifuged once more at 435,400 RCF for 10 minutes. Supernatant was discarded and pellets were used for downstream applications. Density gradient sedimentation: Nuclear extracts were reconstituted in 0% glycerol HEMG buffer (25 mM HEPES pH 7.9, 0.1 mM EDTA, 12.5 mM MgCl2, 100 mM NaCl) supplemented with 1 mM DTT, 1 mM PMSF, and 1x cOmplete protease inhibitor cocktail. 1 mg of nuclear extract was overlaid onto a 10-30% glycerol gradient (in HEMG buffer) prepared in 14 x 89 mm polypropylene centrifuge tubes (Beckman Coulter). Tubes were centrifuged at 274,400 RCF (40,000 RPM), 16 hours, 4C in a SW41-Ti rotor. 500 L fractions were collected and subjected to Western blots directly or after concentrating using 50 L StrataClean Resin (Agilent Technologies). Co-immunoprecipitation: Nuclear extracts were reconstituted in EB300 buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 1 mM MgCl2, 1% NP-40), supplemented with 1 mM DTT, 1 mM PMSF, and 1x cOmplete protease inhibitor cocktail tablet and volume adjusted to 1 mg/ml. Nuclear extracts were incubated overnight with antibodies against BRG1 (Abcam), PBRM1/BAF180 (Cell Signaling Technology 91894), BCL11B (Cell Signaling Technology), RUNX1 (Thermo and Cell Signaling Technology), mouse IgG (Santa Cruz) or rabbit IgG (EMD Millipore). Washed Protein G Dynabeads (Invitrogen) were added to samples for 3 hours. Immunoprecipitants (IPs) were harvested by magnetic separation and remaining ‘supernatant’ was stored as “flow-thru” samples. IP samples were washed 3 times in EB300, resuspended in LDS loading buffer with 100 mM DTT and incubated at 70C for 10 minutes. Dynabeads were removed and remaining samples were subjected to Western blots. Western blotting: Proteins were separated on a 4-12% PAGE gel, transferred onto a PVDF membrane (Millipore), blocked with 5% milk in TBST (20 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween-20) for 1 hour at RT, and incubated overnight at 4C with primary antibodies. Membranes were washed three times with TBST and incubated with secondary fluorophore-conjugated antibodies for 1 hour at RT. Membranes were washed three times with TBST and imaged using Bio-Rad ChemiDoc MP Imaging System.
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