Fig 1: Protein kinase G (PKG) directly binds to HSL for NPR1‐induced activation of HSL. A) Western blotting was used to detect the protein level of HSL in NPR1 overexpressing or knockdown cells. B) Western blots on immunoprecipitation products using anti‐FLAG M2 affinity gel. HEK293T cells were transfected with HSL‐HA/PKG‐FLAG (left) or PKG‐HA/HSL‐FLAG (Right). 48 h later, cell lysates were prepared and used for the studies. C) Coimmunoprecipitation of endogenous PKGα with endogenous HSL from MGC803 and MKN1 cell lysates. D) GST pull‐down assays showing purified GST‐ PKGα binding with purified HSL and endogenous HSL in various cell lysates. E) Immunofluorescence staining showing colocalization of endogenous PKGα/β and HSL in MGC803 and AGS cells. F) Scheme of molecular docking. 3D structures of PKG (below) and HSL (above). G) Diagram for human HSL domain architecture. HSL contains a tissue specific additional N‐terminal domain (1 to 342), an N‐terminal domain (343 to 665), and a regulatory domain N‐terminal kinase domain (666 to 1076). H) Western blots on the input and immunoprecipitation products for interaction between PKGα and FLAG‐tagged truncated HSL. HEK293T cells were similarly transfected/processed, as described in (B). I) Coomassie blue staining of the purified PKGα‐FLAG protein from HEK293T cells and commercially available purified HSL‐HIS protein. J) Western blots on the products from in vitro kinase assays assessing affinity‐purified PKGα phosphorylation of HSL. PKGα proteins were overexpressed/purified using the EZview Red anti‐FLAG M2 affinity gel from the HEK293T cells transiently transfected with pRK5‐PKGα‐FLAG plasmid. K) The migration and invasion abilities of NPR1 overexpressing cells with PKG inhibition. Scale bar = 50 µm. Data presented as mean ± SD, n = 3, p‐values are calculated using two‐way ANOVA tests. * p < 0.05, ** p < 0.01.