Anti sirt2 antibody

Bone marrow-derived dendritic cells were cultured as previously described [19 (link)]. Briefly, bone marrow from the femurs and tibia were collected aseptically from C57BL/6 mice. Cells were cultured for 6 days in RPMI-1640 containing 10% heat-inactivated FBS, 20 ng/ml mGM-CSF (Peprotech), 50 μM β-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated at 5% CO₂ and 37°C. After 3 days, mGM-CSF containing media was supplemented. Loosely adherent cells were collected on the sixth day and used for the further experiment [57 (link)]. Percentage purity of DCs was 65% to 80%.
Total splenocytes were isolated from the spleen of C57BL/6-Tg (TcraTcrb) 1100Mjb/J mice by mechanical disruption. Erythrocytes were lysed by RBC lysis buffer (Sigma) and cells were maintained in RPMI-1640 containing 10% heat-inactivated FBS. Finally, non-adherent cells were collected and were used for mixed lymphocyte proliferation assay.
Anti SIRT2 antibody (Boster, #PA2283), anti NFκB p65 antibody (Cell Signalling, #3034), anti NOS2 antibody (Bimol, #Cay160862), anti Iκα antibody (Santa Cruz Biotech, #A2208) was used for immunoblot and immunofluorescence analysis.

For co-immunoprecipitation, DCs were infected with MOI 10 as mentioned previously. After 10 h post infection, cells were washed with PBS and were lysed in native lysis buffer containing 1% Nonidet P-40, 20 mM Tris (pH 8), 2 mM EDTA, 150 mM NaCl and protease inhibitors mixture (Roche Diagnostics) for 30 min at 4°C. Cell debris was removed by centrifugation at 10,000 rpm for 10 min and the supernatant was treated with anti-SIRT2 antibody (Boster). Antibody-SIRT2 complexes were immunoprecipitated using Protein G linked Sepharose beads (Sigma). Beads were extensively washed with washing buffer and denatured at 95 ^oC for 10 min. Denatured precipitates were subjected to SDS-PAGE (12% gel) followed by transfer to 0.45 μ PVDF membrane. The membrane was blocked using 5% skimmed milk in TTBS for 1h at room temperature. ECL (BioRad) was used for detection and images were captured using ChemiDoc GE healthcare.
All the densitometry analysis was performed using ImageJ platform.