Anti PRDM1 BLIMP1  

Immunohistochemistry of rabbit anti-PRDM1-BLMP1 antibody. Tissue: colon. Fixation: formalin fixed paraffin embedded. Antigen retrieval: not required. Primary antibody: Anti-PRDM1-BLMP1 at 5 μg/mL for 1 h at RT. Secondary antibody: Peroxidase rabbit secondary antibody at 1:10,000 for 45 min at RT. Staining: PRDM1-BLMP1 as precipitated red signal with hematoxylin purple nuclear counterstain.

Immunohistochemistry of rabbit anti-PRDM1-BLMP1 antibody. Tissue: prostate. Fixation: formalin fixed paraffin embedded. Antigen retrieval: not required. Primary antibody: Anti-PRDM1-BLMP1 at 5 μg/mL for 1 h at RT. Secondary antibody: Peroxidase rabbit secondary antibody at 1:10,000 for 45 min at RT. Staining: PRDM1-BLMP1 as precipitated red signal with hematoxylin purple nuclear counterstain.

Western blots using Rockland's affinity purified anti-PRDM1/BLIMP1 antibody show detection of overexpressed PRDM1/BLIMP1 at ~88kDa. Lane 1: mock Raji cell lysate. Lane 2: overexpressed PRDM1/BLIMP1 in whole transfected Raji cell lysate. Lane 3: Raji whole cell nuclear extract. Lane 4: endogenous PRDM1/BLIMP1 in human plasma cell nuclear extract. The identity of the lower dark band at ~50-60kDa is unknown. Primary antibody was used at a 1:1000 dilution in 5% PBS-Tween. Personal communication, Hyesuk Yoon and Jerry Boss, Emory University, Atlanta, GA.

(B？D) ChIP analysis of Blimp-1 binding to the？PD-1？promoter was performed with chromatin from EL4 cells, splenic CD8 T cells, and LCMV antigen-specific transgenic CD8 T cells. Putative Blimp-1 binding sites shown in A were examined for each experimental setting. (B) EL4 cells were transduced with adenovirus expressing GFP or Flag-Blimp-1 and stimulated with PMA/ionomycin before ChIP analysis. Anti-Flag and control mouse monoclonal (HA,12CA5) antibodies were used for the ChIP assays as indicated. (C) Control, unstimulated (day 0, D0) CD8 T cells and anti-CD3/CD28？stimulated splenic CD8 T cells for 5 d (D5) were used for these ChIP assays. Nonspecific rabbit IgG was used as a control ChIP antibody (right). (D) P14 CD8 T cells were adoptively transferred into WT Thy1.2+？C57B/6 mice and analyzed 8 d after infection with LCMV Armstrong. Nonspecific rabbit IgG was used as a control ChIP antibody (right). Results of all ChIP experiments are presented as percentage of input DNA and are representative of three independent experiments (mean ± SD). A two-tailed Student’s？t？test was used to determine significance. **, P < 0.01; ***, P < 0.001. Fig 3. PMID:？24590765

CD8 T cells from LcKO, BcKO, and WT control mice were isolated by magnetic separation and then stimulated with αCD3/CD28 beads for 96 hours. Chromatin was prepared from collected cells and then subject to ChIP. (A) Binding of LSD1 to the CR-B and site-2 regions of the？Pdcd1？locus. (B) Binding of Blimp-1 to site 2 of the？Pdcd1？locus. An α-IgG antibody was used as a control for ChIP experiments. Data are representative of three independent experiments containing 3 to mice per group. A two tailed Student’s？t？test was used to determine statistical significance. **, P < 0.01 ***, P < 0.001. Fig 4. PMID:？31811020

A) C57BL/6 mice were infected with either LCMV Armstrong (Arm) or Clone-13 (Cl-13) for 8 days. CD8 T cells were isolated by magnetic separation and subject to ChIP to assess LSD1 binding at CR-B and site-2 and Blimp-1 binding at site 2 of the？Pdcd1？locus. Naive CD8 T cells were also magnetically isolated (See Methods) from uninfected mice were used as a control, as was an α-IgG antibody.？(B)？Microarray RNA expression data from (40) was analyzed for？Kdm1a？expression (LSD1). (C) Western blot for LSD1 from lysates prepared from CD8 T cells isolated from naive, Armstrong day 8 or Clone-13 day 8 infected C57BL/6 mice. Actin is also shown as a loading control.？(D)？LcKO and littermate control WT mice were infected with LCMV Armstrong. At day 8, gp33-, gp276-, and np396-specific CD8 T cells were isolated, pooled, and subjected to ChIP with α-H3K4me1？and H3K4me2？antibodies at the indicated regions of the？Pdcd1？locus. For (A) and (D), data represent groups of 3 to 4 mice from two independent experiments. A two tailed Student’s？t？test was used to determine statistical significance between samples. *, P < 0.05 ***, P < 0.001. Fig 5. PMID:？31811020