Supplementary Figure 1: Generation and analysis of conditional Pld4 knockout mouse mutation and Pld3 null mutants.

(a) Design of targeting construct in relation to the wild type Pld4 locus, including restriction enzyme sites and location of hybridization probe. FRT recombinase recognition sites flanking the neomycin resistance gene (neo) are indicated with brown triangles and loxP sites are blue triangles. The constructs were designed such that CRE recombinase deletes Pld4 exons 4-6 containing the first HKD domain, and introduces a frame-shift. (b,c) Southern blot analysis of targeted embryonic stem cells, identifying clone 28 as correctly targeted. Experiment was performed twice for each identified cell line. (d) Western blot analysis of mouse PLD4 protein expression. Spleen lysates were analyzed using a rabbit polyclonal anti-PLD4 antiserum. Experiment was performed once. (e) Flow cytometry analysis of PLD4 expression in spleen DCs. DCs (CD11c⁺TCRb^–CD19^–) were gated as shown in the upper panels into pDCs (SiglecH⁺CD45RA⁺), CD8a DCs (CD11b^–CD8a⁺) and CD8^– DCs (CD8^–CD11b⁺) and evaluated for intracellular PLD4 using MAb16 (lower panels) comparing Pld4^fl/fl (red) and Pld4^–/– (blue). Experiment was performed twice. (f) Numbers of sorted DC subsets isolated from spleens from 10 mice of indicated genotype. Error bars indicated standard deviation between three sorts. (g) Elevated MHCII but not CD86 in peritoneal macrophages of Pld4^–/– compared to Pld4^+/– mice n= 6/group. Statistics were determined using a two-tailed Student’s T test assuming equal variance. (h) Proportions of splenic B cell subsets from indicated genotypes. Mice were 8-10 weeks old, 6 mice/group. Marginal zone subset statistics were determined using a two-tailed Student’s T test assuming equal variance. (i) Normalization of MZ B cell frequency in the absence of IFN-γ. n=7,9. Statistics were determined using a two-tailed Student’s T test assuming equal variance. (j-n) Generation and analysis of Pld3 mutants. (j) Exon 9 sequence changes in mutant alleles of Pld3 transmitted by different founder mice. Note some lines had two independent mutant alleles. (k) Immunoblot of PLD3 protein expression in lysates from thioglycolate-elicited macrophages harvested from C57BL/6, Pld4^–/–, or Pld3 Crispr Founder mice. This experiment was performed once. (l-n) Effect of Pld3 or Pld4 genotype on spleen weight (l), splenic NK cell percentage (m), and MHCII expression on resident peritoneal macrophages (n). Pld4^–/– compared to Pld4^fl/fl mice n= 4/group, Pld3^+/– compared to Pld3^–/– n=5/group. Bars show means and standard deviation and each point represents an individual animal. Statistics were determined using a two-tailed Student’s T test assuming equal variance. This experiment was performed twice.