Anti ha agarose

Cells were rinsed with PBS and lysed with buffer A (50 mM HEPES, pH 7.5; 70 mM potassium acetate and 5 mM magnesium acetate) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (10 mM β-glycerophosphate, 1 mM sodium fluoride and 0.1 mM sodium vanadate) for 30 min on ice. Whole cell lysate was cleared by centrifugation for 10 min. Protein concentration was quantified by BCA kit (Thermo Fisher Scientific Inc.). Equal amount of total proteins was incubated with Strep-Tactin resins (IBA GmbH, Goettingen, Germany), ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich), anti-HA agarose (Sigma-Aldrich), normal mouse IgG (Santa Cruz Biotechnology Inc. Dallas, TX, USA), normal rabbit IgG (Santa Cruz Biotechnology Inc.) or antibodies to PYCR1 (Abgent, Inc., San Diego, CA), PYCR2 (LifeSpan BioSciences, Inc. Seattle, WA, USA), RRM2B (Rockland Immunochemicals Inc., Limerick, PA), for three hours to overnight at 4 °C with rocking. Protein A or protein G beads (Santa Cruz Biotechnology Inc.) were added to the antibody-antigen reaction for additional one hour. Beads were collected by centrifugation at 10,000x g for 15 seconds and then washed with ice-cold buffer A three times before boiling in SDS-PAGE sample buffer.

HEK293T cells were grown in 10-cm dishes and transfected with the constructs outlined in the figure legends using either Transit LT1 (Mirus) or calcium phosphate transfection. Sixteen hours later cells were stimulated with IFNα or mock-treated as described in figure legends, then lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.4, 10 mM CaCl₂, 0.1% (v/v) Triton-X, 10% (v/v) glycerol and protease (cOmplete Mini, Roche) and phosphatase inhibitors (PhosSTOP, Roche)) and cleared by centrifugation. Samples were then incubated with 30 μl Protein G Sepharose 4 Fast Flow (GE Healthcare) and anti-STAT1 (Santa Cruz, sc-345) for 6 h, or ANTI-FLAG M2 Affinity Gel (Sigma Aldrich) or Anti-HA Agarose (Sigma Aldrich) and 2 h. Immunoprecipitations were washed 3 times in lysis buffer and bound proteins were eluted by boiling in buffer containing SDS. Samples were then analysed by SDS-PAGE (polyacrylamide gel electrophoresis) and immunoblotting with the stated antibodies.

HEK 293T cells were transfected with 1μg of plasmid expressing CDK11p58-HA using Lipofectamine Plus (Invitrogen) in a 6-well plate and incubated for 24 hours. The cells were then washed with PBS and scraped in the presence of lysis buffer (Cell Signaling) and a protease inhibitor cocktail (cOmplete Protease Inhibitor Cocktail, Roche). The lysate was centrifuged with lysis buffer and 200μg of the total protein was immunoprecipitated with anti- HA agarose (Sigma) for 16 hours. The HA-agarose beads were washed 5 times in PBS and once with in vitro kinase buffer (50 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 1 mM DDT). The resultant immunoprecipitate was analyzed for CDK11p58 kinase activity in in vitro kinase buffer, 50 mM ATP, and SPDEF-GST (2-5 mg/ml). SPDEF phosphorylation was analyzed by 12% SDS polyacrylamide gel and Western blotting using a phospho-serine (pSer) antibody (Chemicon, AB1603). In order to test the inhibitory potential of GADD45, we produced the GADD45 and SPDEF proteins using the TnT^®Quick Coupled Transcription/Translation Systems (Promega) and mixed in the in vitro kinase reaction.

Frozen cell pellets were lysed for 30 min in ice-cold MCLB supplemented with protease and phosphatase inhibitors and cell debris was removed from lysates by centrifugation. The supernatant was subjected to immunoprecipitation with pre-equilibrated anti-HA-agarose (Sigma) overnight at 4°C. Afterwards, agarose beads were washed three times with MCLB buffer and bound proteins were eluted by addition of 4x laemmli buffer and boiling at 95°C for 5 min. Samples were then analyzed by SDS-PAGE and immunoblotting.

For immunopreciputation of ubiquitinated Tsg101 a Flag-tagged Tsg101 was used as described earlier (Dolnik et al., 2010). Cells were transfected with Tsg101-Flag and HA-Ub expression plasmids and 48 h p.tr. lysed in Co-IP buffer (20 mM Tris-HCl, pH 7.5, 100 mM sodium chloride, 0.4% (w/v) deoxycholic acid, 1.0% Triton X-100, 0.5% (w/v) NP-40, 5 mM EDTA and 2% BSA) for 20 min at 4°C. Cell debris were removed by centrifugation at 14.000 rpm for 10 min. Lysates were incubated by with anti-HA agarose or anti-Flag agarose (Sigma-Aldrich) for 3 h at 4°C. Precipitates were washed 3 times with Co-IP buffer without Triton X-100, resuspended in samples buffer boiled and subjected to SDS-PAGE and Western blotting.

The catalytic activity of both PTPN1 and PTPN1 D181A was measured using pNPP. To identify PTPN1 substrates, phosphatase assays were performed with bacterially expressed PTPN1. HA-MLK3 was expressed in 293 T cells, and then purified using anti HA-agarose (Sigma). To purify phospho-MKK7, GST-MKK7 was co-expressed with His-MLK3 in Escherichia coli. To generate phosphorylated JNK, GST-JNK was co-expressed with His-MKK7, (a constitutively activated mutant form of MKK7, harboring Ser-271, Thr-275, and Ser-277 mutations to Glu) in Escherichia coli. Phosphorylated proteins were incubated with purified His-PTPN1 (30 min, 37 °C). Protein phosphorylation levels were detected by immunoblot analysis.