Figure 1
From: MZB1 enables efficient interferon α secretion in stimulated plasmacytoid dendritic cells
MZB1 is expressed in pDCs and inactivation of Mzb1 affects IFNα secretion. (A) Immunoblot analysis of sorted follicular B (FoB) cells (CD19+, AA4.1-, CD23+ CD21+), Marginal Zone B (MzB) cells (CD19+, AA4.1-, CD23-, CD21+) and plasmacytoid dendritic cells (pDCs) (B220+, Siglec H+) to examine levels of MZB1 protein expression in each cell type. GAPDH is used as a loading control. (B) Immunoblot analysis of MZB1 protein level in splenic pDCs at various time points during a 24 h stimulation (image is representative of two experiments) with GAPDH used as a loading control. Uncropped blots are shown in Supplementary Fig. S3A, B. (C) ELISA-based quantification of secreted IFNα and IFNβ levels in the supernatants of 105 pDCs from Mzb1+/+ and Mzb1−/− mice at 24 h post stimulation with CpG A. Each dot represents an individual culture of sorted pDCs derived from the bone marrow of an individual mouse. (D) ELISA-based quantification of secreted TNFα and IL6 levels in the supernatant of 105 sorted pDCs at 24 h post stimulation with CpG B. (E) Secreted IFNα levels, quantified by ELISA, in the supernatant of 105 sorted Mzb1+/+ and Mzb1−/− pDCs at 24 h post stimulation with the TLR7 agonist Imiquimod. ns, non-significant. (F) Flow cytometry analysis of the surface expression of the activation marker CD86 on unstimulated and CpG A-stimulated pDCs. (G) Box plot of quantitative RT-PCR analysis to determine the relative mRNA levels of Ifna in Mzb1+/+ and Mzb1−/− pDCs at 6 and 24 h post stimulation with CpG A, normalized to 18S rRNA levels. The box borders represents the interquartile range and the horizontal line in the box is the median. Numbers of independent samples (n) are indicated below the graph. (H) qRT-PCR analysis of Tnfa (TNFα) and Il6 (IL6) transcript levels in Mzb1+/+ and Mzb1−/− pDCs stimulated with CpG A for 24 h. Transcript levels were normalized to those of 18S rRNA. Error bars show SD. Statistical difference between the mean was analysed by an unpaired two-tailed Student’s t-test. (***) P ≤ 0.0005. ns, non-significant.
