Journal: Nucleic Acids Research

Article Title: The novel GATA1-interacting protein HES6 is an essential transcriptional cofactor for human erythropoiesis

doi: 10.1093/nar/gkad167

Figure Lengend Snippet: GATA1 is a binding partner of HES6 in human erythroid cells. ( A ) Schematic representation of the FLAG-HA tandem affinity purification process. ( B ) A representative image of a whole silver-stained gel of precipitated protein complex samples following FLAG-HA-HES6 tandem affinity purification. Specific bands containing GATA1 protein (∼50 kDa) and HES6 protein (∼35 kDa) are indicated by the arrows. The negative control is cultured human erythroblasts transfected with empty Flag-HA vector (pLV-3*FLAG-HA vector). ( C ) Co-IP results showed that GATA1 or HES6 was immunoprecipitated from primary erythrocyte lysates (at day 9) with an anti-GATA1 or anti-HES6 antibody. GATA1 or HES6 in the immunoprecipitate was detected with an anti-GATA1 or anti-HES6 antibody, respectively. ( D ) Co-IP results confirmed that the HES6 and GATA1 interaction occurred in the nucleus but not in the cytoplasm. Total proteins were used as a positive input control. ( E ) Representative western blotting analysis of the pull-down assay using purified GST–GATA1 with purified His-HES6, and purified GST–HES6 with purified His-GATA1. ( F ) Schematic diagram showing the functional domains of HES6 and GATA1, respectively. The HES6 protein includes a bHLH, Orange, PEST and WRPW domain. The GATA1 protein includes N-terminal (N-TAD) and C-terminal (C-TAD) domains and two zinc finger (NF and CF) domains located in the middle of the molecule. ( G ) Left panel: purified GST–HES6 deletion mutants were incubated with HEK293T cell lysates expressing Flag-tagged GATA1, and their interactions were analyzed using a pull-down assay. Right panel: EGFP-tagged GATA1 deletion mutants and Flag-tagged HES6 were co-transfected into HEK293T cells. Their interactions were analyzed using a Co-IP assay. ( H ) Under conditions of equivalent amounts of transfected plasmids and streptavidin-coupled Dynabeads, the DNA pull-down assay was used to evaluate the alteration in interactions between exogenous Flag-GATA1 and bio- AHSP (left panel) and bio- PRG2 (right panel) promoter DNA in the presence or absence of exogenous Flag-HES6. The expression of Flag-HES6 and Flag-GATA1 in protein supernatants served as loading controls. The empty pCMV3-C-flag vector was used as a negative control.

Article Snippet: DNA was then purified using column-based DNA purification kits (Sangon Biotech Co., Shanghai, China, #B110093-0100).

Techniques: Binding Assay, Affinity Purification, Staining, Negative Control, Cell Culture, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Pull Down Assay, Purification, Functional Assay, Incubation, Expressing