Journal: The Journal of Immunology Author Choice

Article Title: The Zinc Finger Protein Zbtb18 Represses Expression of Class I Phosphatidylinositol 3-Kinase Subunits and Inhibits Plasma Cell Differentiation

doi: 10.4049/jimmunol.2000367

Figure Lengend Snippet: Zbtb18 directly binds and suppresses expression of PI3K class I genes. ( A ) Splenocytes were transduced with control (RFP + ) or Zbtb18 (GFP + ) vectors. Cells were analyzed by flow cytometry and mixed to achieve a 1:1 ratio of B220 + -GFP + – and RFP + -expressing cells, followed by stimulation with anti-CD40 and IL-4. Two days after stimulation, RFP + or GFP + B220 + CD138 – live cells were sorted by flow cytometry and subjected to Chip qPCR analysis of the ATAC-seq differential peaks of Pik3r1 , Pik3r5 , and Pik3r6 genes. Data are from pooled samples from three independent experiments performed (total n = 3). ( B ) De novo motif–enrichment analysis in repressed ATAC-seq peaks of Zbtb18 versus control (Ctrl) cells. Motif density distribution relative to the peak summit for Zbtb18, Pu.1 and Oct2 motifs in the repressed ATAC-seq peaks (bottom). ( C ) Chip qPCR analysis of interactions between Myc-tagged Zbtb18 (WT), or ΔZF with the indicated genes. Ctrl B cells transduced with empty GFP-expressing vector. Conserved Zbtb18-binding motif identified in the ATAC-seq peaks of the indicated genes are shown in red. Analysis was performed on transduced sorted cells (live B220 + GFP + cells) 2 d after anti-CD40 and IL-4 stimulation. Data show pooled samples collected in three independent experiments performed (total n = 3, biological replicates). ( D ) Immunoblot analysis of p85a, p101, and GAPDH in Ctrl/RFP– or Zbtb18/GFP–transduced B cells 2 d after being cocultured with or without anti-CD40 and IL4 stimulation (left) [as in (A) above]. Cells were sorted prior to analysis on day 2 (gated on live B220 + CD138 − GFP + or RFP + cells). Quantification of p85a or p101 expression normalized with GAPDH (right). Figure shows combined data collected in two independent experiments performed. Each circle represents data from one experiment. ( E ) qPCR analysis of the indicated genes in control- and Zbtb18-transduced B cells, sorted (as GFP + B220 + CD138 − live cells) 2 d poststimulation with anti-CD40 and IL-4. Expression of mRNA is presented relative to the abundance of GAPDH. Data in (E) show the results from one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The whole-cell lysis or immunoprecipitated fraction was separated by SDS-PAGE and transferred onto nitrocellulose membranes (162-0115; Bio-Rad Laboratories), immunoblotted with anti-p85 Ab (ABS234; MilliporeSigma), anti-p101 Ab (D32A5, no. 5569; CST), or anti-GAPDH (6C5, MAB374; MilliporeSigma), anti–p-AKT (Ser473; New England Biolabs), anti-AKT (pan) (C67E7; CST), anti-Myc (Ab9132; Abcam), or anti-GAPDH (6C5, MAB374; Merck Life Science).

Techniques: Expressing, Transduction, Control, Flow Cytometry, ChIP-qPCR, Plasmid Preparation, Binding Assay, Western Blot