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F., Macara I. E3 ligase activity (9). In addition, c-IAP1/2 was reported to act as an E3 ligase for RIP1 and (11) recently provided evidence showing that TRAF2 actually is an E3 ligase for RIP1 Lys-63 polyubiquitination were generous gifts (17) and were amplified and used for expression in HEK293 and HeLa cells. RSK1-HA, HA-Lys-48-ubiquitin, and HA-Lys-63-ubiquitin were purchased from Addgene (Cambridge, MA). GST-IB was purified as described previously (16). and mutants were constructed from using a site-directed mutagenesis kit (Stratagene, La Jolla, CA). RSK2 was subcloned into the vector by Xba1,BamH1 from the mutant was constructed as described above. Lentivirus plasmids containing (#1, TRCN0000004572;#2, TRCN0000004574), (TRCN0000003783), and (TRCN0000003778) were purchased from Thermo Scientific (Huntsville, AL). Cell Culture and Transfection TRAF2+/+ and TRAF2?/? MEFs were a generous gift (17). They were cultured with DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37 C in a 5% CO2 incubator. Human embryonic kidney (HEK293) cells, 293T cells, and HaCaT cells were grown in Dulbecco’s modified Eagle’s medium (Hyclone, San Diego, CA) supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA), 100 units/ml penicillin, and 100 mg/ml streptomycin and cultured at 37 C in a humidified incubator with 5.0% CO2. HeLa cells (human cervix adenocarcinoma) were grown in Eagle’s minimum essential medium (MEM) supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin and cultured at 37 C in a humidified incubator with 5% CO2. The cells were UNC0379 maintained by splitting at 90% confluence, and media were changed every 3 days. When cells reached 50C60% confluence, transfection was performed using JetPEI (Polyplus-transfection Inc., New York, NY) following the manufacturer’s suggested protocol. The cells were cultured for 36C48 h, and then proteins were extracted for further analysis. Lentiviral and Retroviral Infection To construct knockdown TRAF2, RSK2, and cIAP1/2 cells, the lentivirus plasmid of was co-transfected into 293T Mouse monoclonal to CD81.COB81 reacts with the CD81, a target for anti-proliferative antigen (TAPA-1) with 26 kDa MW, which ia a member of the TM4SF tetraspanin family. CD81 is broadly expressed on hemapoietic cells and enothelial and epithelial cells, but absent from erythrocytes and platelets as well as neutrophils. CD81 play role as a member of CD19/CD21/Leu-13 signal transdiction complex. It also is reported that anti-TAPA-1 induce protein tyrosine phosphorylation that is prevented by increased intercellular thiol levels cells UNC0379 together with and viral supernatant fraction. At 3C4 days after infection, the appropriate experiments were performed using these cells. For generation of stable HaCaT cells expressing or plasmid was co-transfected into 293T cells together with and viral supernatant fractions were infected into HaCaT cells together with 10 g/ml Polybrene. At 16 h after infection, the medium was replaced with fresh medium containing 1 g/ml puromycin, and cells were incubated for 6 days. Immunoblotting and Immunoprecipitation Protein samples from cells were extracted with Nonidet P-40 cell lysis buffer (50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 0.5% Nonidet P-40, and protease inhibitor mixture). For immunoblotting, 30 g of protein were used with appropriate specific antibodies and an alkaline phosphatase (AP)-conjugated secondary antibody, and proteins were detected by the STORM machine using the UNC0379 fluorescence/chemiluminescence mode (Amersham Biosciences). For immunoprecipitation, the extractions were combined with agarose A/G beads (50% slurry) by rocking at 4 C overnight. The beads were washed, mixed with 6 SDS sample buffer, boiled, and then resolved by 10% SDS-PAGE. The proteins were detected using the appropriate specific antibodies and an AP-conjugated secondary antibody. For immunoprecipitation (IP) under denaturing conditions, proteins were extracted using regular IP buffer plus 1% SDS and heated at 95 C for 5 min. The samples were diluted 1:10 in regular IP buffer before IP. The beads were washed, mixed with 6 SDS sample buffer, boiled, UNC0379 and then resolved by 10% SDS-PAGE. The proteins were visualized by immunoblotting. Immunofluorescence Staining Appropriate cells were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 for 30 min. Fixed cells were then incubated with an RSK2 mouse monoclonal antibody (Santa Cruz Biotechnology) and TRAF2 rabbit polyclonal antibody (Cell Signaling Biotechnology, Inc.) overnight followed by incubation with red fluorescent Alexa Fluor 568 dye-labeled anti-mouse IgG or green fluorescent Alexa Fluor 488 dye-labeled.