Anti risp

CD4⁺ Foxp3-YFP⁺ CD25⁺ cells were collected using fluorescence-activated cell sorting. Following isolation, cells were lysed in cell lysis buffer (Cell Signaling), resolved on 12% polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes. Membranes were blocked using 5% milk for 1 hour and then incubated with anti-RISP (Mitosciences, catalog no. ab14746; used at 1:250 dilution) and anti-β-actin (Sigma, catalog no. T9026; used at 1:2000 dilution) overnight. Membranes were washed with TBST the next day and then incubated with goat anti-mouse IRDye 680RD (Li-cor, catalog no. C50721, 1:5,000 dilution) and goat anti-rabbit IRDye 800CW (Li-cor, catalog no. C60321; 1:10,000 dilution) for one hour. Following another TBST wash the membranes were directly imaged using the Odyssey Fc Analyzer (Li-cor).

Lineage negative cells were purified following manufacturer's instruction (Stem Cell Technologies). Then, cells were lysed in cell lysis buffer (Cell Signaling), resolved on 4 to 20% polyacrylamide gels (BioRad) and transferred to nitrocellulose membranes. Membranes were blocked using 5% Milk for 1 hour and then incubated in the primary antibody overnight. Antibodies used were anti-RISP (Mitosciences; Catalog Number: ab14746. Used at a 1:500 dilution), anti-TFAM (a gift from Gerald Shadel, Yale University. Used at a 1:500 dilution), anti-β-actin (Sigma; Catalog Number: A2066. Used at 1:10,000 dilution) and anti-α-Tubulin (Sigma; Catalog Number: T9026. Used at a 1:2000 dilution). Membranes were washed 4 times with TBST and then incubated with secondary antibody. Secondary antibodies used were Anti-mouse IgG, HRP-Linked (Cell Signaling; Catalog Number: 7076S. 1:10,000 Dilution, used for anti-TFAM), Anti-rabbit IgG, HRP-Linked (Cell Signaling; Catalog Number: 7074S. 1:10,000 Dilution, used for anti-β-actin), and Goat Anti-Mouse IRDye 680RD (Li-cor; Catalog Number: C50721. 1:5,000 dilution. Used for anti-RISP and Anti-Tubulin). TFAM and actin blots were then treated wth ECL (Pierce) and developed using film. The RISP and tubulin blots were directly imaged using the Odyssey Fc Analyzer (Licor).