Hrp conjugated secondary antibody

Thymocytes isolated from WT or R360P mice were washed with PBS and rested for 30 min at 37°C. Cells were left unstimulated or stimulated with anti-CD3 (clone 2C11) at 37°C over time as described in each experiment. Peripheral CD8-OTI T cells were isolated from WT or R360P-OTI mice by negative depletion, incubated with OVA or G4 peptide tetramers (NIH Tetramer Core Facility) in PBS for 15min on ice, and transferred to 37°C for stimulation over time as described. Cells were lysed with lysis buffer with a final concentration of 1% NP40 (containing inhibitors of 2 mM NaVO4, 10 mM NaF, 5 mM EDTA, 2 mM PMSF, 10 μg/ml Aprotinin, 1 μg/ml Pepstatin and 1 μg/ml Leupeptin). For cell line experiments, HEK293 cells were harvested and lysed 24 hours after transfection.
Lysates were placed on ice and centrifuged at 13,000 × g to pellet cell debris. Supernatants were run on NuPAGE 4%–12% Bis-Tris Protein Gels (Thermo Fisher Scientific) and transferred to PVDF membranes. Membranes were blocked using TBS-T buffer containing 3% BSA, and probed with primary antibodies as described in each experiment, overnight at 4°C. The following day blots were rinsed and incubated with HRP-conjugated secondary antibodies (Southern biotech). Blots were developed using a chemiluminescent substrate and a BioRad Chemi-Doc imaging system (Bio-Rad).

Dox-inducible cell lines were treated with 1 μg/ml of dox for 5 days and cell lysates were prepared with lysis buffer (50 mM Tris pH 7.6, 150 mM NaCl, 3 mM MgCl, 10% glycerol, 0.5% NP-40) and quantified by Bradford assay. Equivalent amounts of each sample were resolved by SDS-PAGE and Western blotted with antibodies against Xrn1 (Bethyl; diluted 1:200), Dis3L2 (kindly provided by Torben Jensen; diluted 1:500), Ccr4 (diluted 1:1000), Pan2 (diluted 1:1000) and actin (diluted 1:200). Primary antibodies were followed by HRP-conjugated secondary antibodies (Southern Biotechnology, 1:5000).