Fig. 2: KIF7 mediates interaction between P4HA2 and the Hedgehog components. | Leukemia

Fig. 2: KIF7 mediates interaction between P4HA2 and the Hedgehog components.

From: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

Fig. 2

A Schematic of full-length KIF7 proteins. The KIF7 protein encodes a 1343 aa protein with a kinesin motor ___domain, GLI-binding ___domain (BD), Nephoronophthisis-1-interacting ___domain (NPHP1-ID) and a Structural Maintenance of Chromosomes (SMC) ___domain representing the ATPase activity. Various truncated mutation constructs of KIF7-Flag are shown schematically. B Mapping the interaction ___domain of KIF7 with P4HA2. HEK293T cells were transfected with constructs shown as (A). Cell lysates were prepared and subjected to immunoprecipitation (IP) with M2 anti-Flag beads. C Mapping the minimum interaction ___domain of KIF7 with P4HA2. HEK293T cells were transfected with constructs “a”, “f” and “g” mutants. Cell lysates were prepared and subjected to Flag IP. D, E Endogenous co-immunoprecipitated P4HA2 and KIF7 interact with each other separately in HEK293T cells. Endogenous KIF7 and P4HA2 were precipitated from a HEK293T cells extract and analyzed by immunoblotting. F P4HA2 interacts with SUFU and GLI2. HEK293T cells were transfected with Flag-tagged P4HA2. Cell lysates were subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag, KIF7, SUFU and GLI2 antibodies. G P4HA2 interacts with GLI1. HEK293T cells were transfected with Flag-tagged P4HA2 and V5-tagged GLI1. Cell lysates were subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag, KIF7 and V5 antibodies. H P4HA2 directly interacts with KIF7 in vitro. His-tagged KIF7, GLI1, SUFU, and Flag-tagged P4HA2 proteins were purified and incubated. After His pulldown, the complex was analyzed by WB. I KIF7 knockdown decreases the interaction between P4HA2 with SUFU. IgG-P4HA2 was overexpressed in shKIF7-1 and shKIF7-2 HEK293T cells. Cell lysates were subjected to IgG IP. Co-immunoprecipitated SUFU was detected by WB. J KIF7 knockdown decreases the interaction between P4HA2 and GLI1. IgG-P4HA2 and V5-GLI1 were co-overexpressed in KIF7 stable knockdown HEK293T cells (shKIF7-1 and shKIF7-2). Cell lysates were subjected to IgG IP. Co-immunoprecipitated V5-GLI1 was detected by WB.

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