Anti blimp1

HEK 293T cells were transiently transfected alone or in combination with expression plasmids coding for ER-tagged NFATc1/A and NFATc1/C or Flag-tagged Blimp-1 and its mutants (Figure 2F) (30 (link), 47 (link)). Cells were lysed in IP lysis buffer (Thermo Scientific) and CoIP was performed as described earlier (30 (link)), using anti-ER (Santa Cruz) and anti-Flag (M2, Sigma) Abs. For CoIP of Blimp-1 and NFATc1 from primary T cells, 1x10⁷ tTreg and Tconv cells were activated and expanded with anti-CD3/CD28 beads (Invitrogen) for 7 days. CoIP was performed with the Nuclear Complex Co-IP Kit (Active motif) using anti-Blimp-1 (C14A4, cell signaling) and anti-NFATc1 (7A6, BD Pharmingen) Abs.

Recombinant human M-CSF was purchased from R&D Systems (Minneapolis MN, USA). RANKL was obtained from PeproTech EC (London, UK). CsA was purchased from Calbiochem (La Jolla CA, USA) and poly I:C was from Sigma-Aldrich (St. Louis MO, USA). Primary antibodies used in the study included monoclonal anti-FLAG, anti-Sirt6 (Sigma-Aldrich), anti-Blimp1 (Cell Signaling Technology, Beverly MA, USA), anti-V5 (Invitrogen, Carlsbad CA, USA), anti-MafB (Novus Biologicals, Littleton CO, USA), anti-NFATc1 and anti-GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz CA, USA) followed by secondary horseradish peroxidase-conjugated antibody. Anti-Atp6v0d236 (link) antibody was kindly provided by Y. Choi (University of Pennsylvania, Philadelphia PA, USA). The pCDH-3x-Flag-Sirt6 and pcDNA 3.1-V5-Sirt6 were made as described previously21 (link). For retroviral expression, the Flag-Sirt6 DNA was subcloned into pMX-puro to make pMX-puro-Flag-Sirt6. A retroviral vector, pMX-puro was provided by Dr T Kitamura (University of Tokyo, Tokyo, Japan). The pMX-puro-Flag-Blimp1 plasmid12 (link) was provided by J. Rho (Chungnam National University, Daejeon, Korea). The retroviral vector containing a constitutively active form of NFATc1 (caNFATc1) was previously described37 (link).

Cell lysates were harvested in Sumo lysis buffer including protease inhibitors (Roche) as described previously [96 (link)]. Protein concentration was determined using the Sumo protein assay (Biorad), and proteins were separated in SDS-10% polyacrylamide gels and then transferred onto a nitro-cellulose membrane. Membranes were blocked in PBS containing 5% milk, and 0.1% Tween 20 solution. Membranes were then incubated in the following primary antibodies: anti-Z (Santa Cruz, product # sc-53904, 1:250), anti-BMRF1 (Millipore, product # MAB8186, 1:3,000), anti-R rabbit polyclonal antibody directed against the R peptide (peptide sequence EDPDEETSQAVKALREMAD, 1:2,500), anti-KLF4 (Cell Signaling, product # 4038, 1:1,000), anti-BLIMP1 (Cell Signaling, product # 9115, 1:1,000), anti-β-actin (Sigma, product # A5441,1:5,000), anti-tubulin (Sigma, product # T5168, 1:2,000), and anti-involucrin (Sigma, product # I9018, 1:3000). The secondary antibodies used were horseradish peroxidase (HRP)- labelled goat anti-mouse antibody (Fisher Scientific, 1:5,000) and HRP- labeled anti-rabbit antibody (Fisher scientific, 1:5,000).