Fig 1: PKG phosphorylates HSL at S855 and S951 for NPR1‐induced activation of HSL. A) Coomassie blue staining of the commercially available purified PKGα‐GST protein and HSL‐HIS protein. B) Phosphorylation sites identified by LC‐MS/MS. C) Phosphorylation sites of PKG on HSL, Phosphorylation modification sites detected by LC‐MS using in vitro kinase reaction products. the reaction products (left), specific phosphorylation sites predicted by iGPS 1.0 software (right). D) Western blotting was used to confirm the phosphorylation of HSL by commercially available purified PKGα‐GST protein in vitro kinase assay product. E) Western blotting was used to confirm the phosphorylation level of HSL in NPR1 overexpressing or knockdown cells. F) Western blots on WT and HSL‐KO AGS cell lysates. G) Western blotting was used to detect the protein expression efficiency of NPR1, HSLWT, and HSLS855A/S951A in two AGS HSL‐KO cells. H) The migration and invasion abilities of NPR1 overexpressing AGS HSL‐KO cells with HSLWT or HSLS855A/S951A overexpression. Scale bar = 50 µm. Data presented as mean ± SD, n = 3, p‐values are calculated using two‐way ANOVA tests. I) Staining of lipid droplets in NPR1 overexpressing AGS HSL‐KO cells with HSLWT or HSLS855A/S951A overexpression. Cells were loaded with oleic acid (50 µm) to increase the intracellular LD content before DNA transfection. Data presented as mean ± SD, n = 3, p‐values are calculated using two‐way ANOVA tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 2: 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.
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