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. Author manuscript; available in PMC: 2019 Aug 6.
Published in final edited form as: Cell Rep. 2019 Jul 30;28(5):1307–1322.e8. doi: 10.1016/j.celrep.2019.06.079

Figure 3. FBXO11 Supports CD40 Expression by Targeting the Repressor CTBP1.

Figure 3.

(A) Immunoblot analysis of CD40, poly-ubiquitin (poly-Ub), or control GAPDH levels in WCE from Cas9+ Daudi B cells that expressed the indicated sgRNA, treated with DMSO or the proteasome inhibitor bortezomib (200 nM) for 16 h. Increased poly-Ub signal indicates on-target bortezomib activity.

(B) qRT-PCR analysis of CD40 mRNA abundances relative to 18S control levels in Cas9+ Daudi B cells expressing the indicated sgRNA.

(C) FACS analysis of PM CD40 abundances in Cas9+ Daudi B cells expressing the indicated control or FBXO11 targeting sgRNA as well as the indicated control or CD40 cDNA construct.

(D) FACS analysis of Daudi B cell PM CD40 MFI as in (C) from n = 3 replicates.

(E) FACS analysis of PM Fas abundances in Cas9+ Daudi B cells expressing the indicated control or FBXO11 targeting sgRNA as well as the indicated control or CD40 cDNA rescue construct, stimulated by Mega-CD40L (50 ng/mL for 48 h), as indicated.

(F) Mean + SD Log2-normlized CTBP1 sgRNA abundances from both pre-FACS sort input libraries and from all four screen replicates are shown.

(G) Immunoblot analysis of CTBP1, FBXO11, and GAPDH control abundances in WCE from Cas9+ Daudi B cells expressing the indicated control, FBXO11, or either of two independent CTBP1 targeting sgRNAs.

(H) PM CD40 abundances in Cas9+ Daudi B cells expressing the indicated two sgRNAs.

Mean + SD levels from at least n = 3 experiments and shown in (B), (D), and (H). *p < 0.05, **p < 0.01, ***p < 0.001, ns, non-significant. Immunoblot results were representative of n = 3 experiments.

See also Figure S3.