S protein agarose

NETN buffer (20 mM Tris–HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40) was used to lyse HeLa cells or 293T cells by rotation at 4°C for 20 min. After the removal of cell debris by centrifugation (14 000 rpm for 10 min), the soluble fractions were collected and incubated with S protein agarose (Merck-Millipore) for 3 h at 4°C. S protein beads were washed three times with NETN buffer and boiled with 2× SDS loading buffer at 100°C for 8 min. The samples were then subjected to SDS-PAGE and immunoblotting with specific antibodies.

293T cells transfected with HA-empty vector, HA-Casp8p41, HA-Casp8p41 V150E/L157K, or HA-Casp8p41 V150A/L157A and pSPN-empty vector or pSPN-Bak expression vector for 24 h were collected, washed, and lysed. Cells were lysed in 100 µl of lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin, and 1 mM PMSF) for 10 min on ice. Cells were then centrifuged at 400 g, for 5 min, at 4°C. For cell fractionation, the lysate was then further centrifuged at 15,000 g for 5 min at 4°C, resulting in a mitochondrial enriched pellet and cytosolic supernatant. Aliquots containing 900 µg of protein were incubated with 12 µl of anti-HA affinity matrix (Roche) or S-protein agarose (EMD Millipore) for 4 h at 4°C. After beads were washed twice with 150 µl TBST, bound protein was eluted. Samples were run on SDS-polyacrylamide gels and transferred to PVDF membranes for 2 h at 1,200 mAmps in transfer buffer (24 mM Tris and 192 mM glycine). The membranes were then blocked in TBST (20 mM Tris, 150 mM NaCl, and 0.05% Tween-20, pH 7.4) containing 2% bovine serum albumin (Sigma-Aldrich) for >1 h at 21°C or overnight at 4°C followed by immunoblotting, without stripping between blots. Primary antibodies used were: anti-HA peroxidase high-affinity 3F10 (rat; Roche) or anti–S-Peptide HRP (mouse; EMD Millipore).

Tandem affinity purification (TAP) was carried out as described previously (43 (link)). Briefly, S-protein-2xFLAG-Streptavidin binding peptide (SFB)-tagged TLK1, SFB-TLK1 1–240, SFB-TLK1 D607A, SFB-TLK1 133–208, SFB-TLK1 133–208 Y149A F150A, or SFB-TLK2 were transfected into HEK293T cells. Cells were harvested 24 hours after transfection using NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.5% NP-40) supplemented with 2 μg/mL aprotinin (Thermo, AAJ60237MB) and 5 μg/mL pepstatin A (Thermo, PI78432) at 4°C for 20 minutes. Lysates were centrifuged at 9,000 × g, 4°C for 20 minutes to yield supernatant as soluble fraction. The pellet was washed 1x with PBS and centrifuged at 9,000 × g, 4°C for 5 minutes and lysed with chromatin extraction buffer (NETN buffer, 10 mM MgCl₂, and Turbonuclease, Accelagen, N0103M) at 4°C for 1 hour followed by centrifugation at 9,000 × g, 4°C for 20 minutes to obtain chromatin fraction. Both soluble and chromatin fractions were incubated with streptavidin sepharose (200 μl) (GE Healthcare, GE17–5113-01) at 4°C for 1 hour followed by washing with NETN buffer three times. The protein complexes were eluted with 2 mg/mL biotin at 4°C for 1 hour. The eluents were then incubated with S-protein agarose (EMD Millipore, 69704–3) overnight at 4°C, washed three times with NETN buffer, and eluted in 1x Laemmli buffer.