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. 2021 Sep 30;297(5):101264. doi: 10.1016/j.jbc.2021.101264

Figure 2.

Figure 2

Hop2 interacts with adipocyte CEBP.A, schematic illustration of CEBPα as a representative of adipocyte CEBPs (α, β, and δ) and Hop2 primary structure. Amino acid residues are indicated by numeric letters. B, pull-down assays for a direct interaction between bacterially expressed GST-Hop2 and His-CEBPα fusions. Purified His-CEBPα was incubated with GST-Hop2 or GST-bound glutathione agarose. The proteins retained were resolved with SDS-gel and visualized by Coomasie blue staining. C, pull-down assays. Ni-affinity beads were incubated with a mixture of His-CEBPα and GST-Hop2 or GST-Hop2 alone (negative control). The bound proteins were resolved with SDS-gel and visualized by Coomasie blue staining. D, coimmunoprecipitation (co-IP) assays for the interaction between HA-tagged Hop2 and Flag-tagged CEBPα. Nuclear extracts (NEs) from COS1 cells transfected with HA-Hop2 and/or with anti-HA or anti-Flag antibodies (Abs). Western blot analysis of precipitates for Flag and HA (top two panels) and NE input (10%) for each co-IP (bottom panel). E, co-IP and Western blot analysis of NEs from wt mouse visceral adipose tissue (VAT) for endogenous interaction (top two panels) and expression of Hop2 and CEBPα (bottom two panels). Abs used for IP and IB are indicated. F, co-IP and Western blot analysis of NEs from wt mouse VAT for endogenous interaction (top two panels) and expression of Hop2 and CEBPβ (bottom two panels). Abs used for IP and IB are indicated. G, co-IP and Western blot analysis of NEs from wt mouse VAT for endogenous interaction (top two panels) and expression of Hop2 and CEBPδ (bottom two panels). Abs used for IP and IB are indicated. H, immunofluorescence for colocalization of Hop2 and CEBPα in nuclei of adipocytes. Sections of paraffin-embedded mouse adipose tissues were stained with Abs indicated on the top left of each image. Nuclei are revealed with Hoechst staining. AD, activation domain; CC, coiled-coil motif; CEBP, CCAAT enhancer binding protein; DBD, DNA-binding domain; GST, glutathione-S-transferase; LZ, leucine zipper domain; NLS, nuclear location signal (EWRKRKR).