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. Author manuscript; available in PMC: 2017 Nov 3.
Published in final edited form as: Mol Cell. 2016 Oct 20;64(3):507–519. doi: 10.1016/j.molcel.2016.09.010

Figure 3. Arg97 and Leu99 in SLBP are necessary for its interaction with cyclin F.

Figure 3

(a) Schematic representation of SLBP highlighting three putative CY motifs in SLBP.

(b) HEK293T cells were transfected with either an empty vector (EV) or FLAG-STREP-tagged SLBP constructs. Cells were treated with MLN4924 for four hours prior to collection. Whole cell extracts were affinity purified with anti-STREP resin and immunoblotted as indicated.

(c) HEK293T cells were transfected with FLAG-tagged SLBP constructs. Whole cell extracts were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were then immunoblotted as indicated.

(d) HEK293T cells were transfected with either EV or FLAG-tagged SLBP constructs. Cell lysates were immunoprecipitated with anti-FLAG resin and bound RNA was purified. Random primed cDNAs were prepared from equal volume of isolated RNA and analyzed by qPCR for total HIST1H3H mRNA and total H2AFX mRNA. The data are presented as fold change relative to the EV sample.

(e) HEK293T cells were transfected with FLAG-STREP-tagged SLBP constructs. Cells were treated with MLN4924 for four hours prior to collection. Whole cell extracts were affinity purified with anti-STREP resin and immunoblotted as indicated.

(f) Alignment of the CY motif in SLBP orthologs. Critical amino acids required for cyclin F (RxL/I) and cyclin A2 (RKL/IL) binding are highlighted in gray.

See also Figure S3