binding affinity measurement of dusp18 and human gpr54 ct  (CH Instruments)

Journal: Nature Communications

Article Title: Kisspeptin-10 binding to Gpr54 in osteoclasts prevents bone loss by activating Dusp18-mediated dephosphorylation of Src

doi: 10.1038/s41467-024-44852-9

Figure Lengend Snippet: a – d Anti-Gpr54 IP was performed on WCL derived from RAW264.7 cells treated with indicated dose of Kp-10 for 20 min. Beads were analyzed by phosphatase activity assay ( a ), mass spectrometry assay ( b ), IB analysis ( c ) and quantification of protein levels ( d ). e , f Protein co-localization was analyzed by IF staining in primary pre-osteoclast with 10 nM Kp-10 treatment for 20 min. Representative images of endogenous Gpr54 and Dusp18 were shown by TIRF microscopy ( e ), and MOC was used to quantify the degree of colocalization in cells from multiple images taken randomly ( f ). Scale bar, 50 µm. g , h binding affinity was measured by SPR binding analysis. The binding affinity of DUSP18 and human GPR54 CT was measured at 40 nM, RUmax = 68.22, Chi = 1.01 ( g ), DUSP18 and mouse Gpr54 336–342 was measured at 1.8 µM, RUmax = 154.6, Chi = 3.42 ( h ). i – l IB and quantification of protein levels analysis of total samples and GST pull-downs using GST proteins including GST, GST-GPR54 CT, GST-GPR54 CT m1 (R336A&P339A), GST-GPR54 CT m2 (delta 339–344) incubated with WCL of 293 T cells transfected with DUSP18-HA ( i ) and quantification of protein levels ( j ), Total samples and GST pull-downs using His-SRC protein (0 or 0.1 μg) purified from Sf9 cells, His-DUSP18 (0, 0.1 or 0.2 μg), and GST proteins purified from E. coli ( k ) and quantification of protein levels ( l ). Data represent means ± SEM. P values were determined by one-way ANOVA analysis ( a , d , j , l ) or two-tailed Student’s t -test ( f ). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The binding affinity of DUSP18 and human GPR54 CT was measured at 40 nM, RUmax = 68.22, Chi = 1.01 ( g ), DUSP18 and mouse Gpr54 336–342 was measured at 1.8 μM, RUmax = 154.6, Chi = 3.42 ( h ). i – l IB and quantification of protein levels analysis of total samples and GST pull-downs using GST proteins including GST, GST-GPR54 CT, GST-GPR54 CT m1 (R336A&P339A), GST-GPR54 CT m2 (delta 339–344) incubated with WCL of 293 T cells transfected with DUSP18-HA ( i ) and quantification of protein levels ( j ), Total samples and GST pull-downs using His-SRC protein (0 or 0.1 μg) purified from Sf9 cells, His-DUSP18 (0, 0.1 or 0.2 μg), and GST proteins purified from E. coli ( k ) and quantification of protein levels ( l ).

Techniques: Derivative Assay, Phosphatase Assay, Mass Spectrometry, Staining, Microscopy, Binding Assay, Incubation, Transfection, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: Kisspeptin-10 binding to Gpr54 in osteoclasts prevents bone loss by activating Dusp18-mediated dephosphorylation of Src

doi: 10.1038/s41467-024-44852-9

Figure Lengend Snippet: a The binding affinity of DUSP18 and Src was measured at 5.9 nM (RUmax = 130.6, Chi = 3.42) by SPR binding analysis. b – f IB and quantification of protein co-IP levels. Anti-Flag IP derived from 293 T cells transfected with HA-Src and either DUSP18-Flag or DUSP18 (C104S)-Flag constructs with or without Kp-10 treatment for 20 min were lysed and subjected to anti-FLAG IP ( b ). Phosphatase reaction products using His-DUSP18 and His-DUSP18 (C104S) proteins purified from E. coli and SRC proteins purified from Sf9 insect cells ( c ) and quantification of protein levels ( d ). Anti-Src IP derived from RAW264.7 cells treated with or without Kp-10 treatment for 20 min were lysed and subjected to anti-Src IP ( e ) and quantification of protein levels ( f ). g , h Src and Dusp18 co-localization was analyzed by IF staining in primary pre-osteoclasts treated with 10 nM Kp-10 treatment for 20 min. Representative images of endogenous Dusp18 and Src were shown by TIRF microscopy ( g ), and MOC was used to quantify the degree of colocalization in cells from multiple images taken randomly ( h ). Scale bar, 50 µm. i – m IB and quantification of protein levels analysis. Anti-Flag IP derived from 293 T cells transfected with HA-Src, GPR54-myc, and either DUSP18-Flag or DUSP18 (C104S)-Flag constructs after Kp-10 treatment for 20 min ( i ) and quantification of protein levels ( j , k ). WCL derived from WT and Dusp18 −/− BMMs treated with or without Kp-10 for 20 min ( l ) and quantification of protein levels ( m ). n , o WT and Dusp18 −/− BMMs were cultured in vitro on dentin slices, and after incubation with M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 5–7 days, the pits formed by osteoclast absorption activity were scanned by confocal microscopy (XY and z sections). Scale bars, 125 µm ( n ), and pits depth were quantified by confocal microscopy ( o ). Data represent means ± SEM. P values were determined by one-way ANOVA analysis ( d , f , j , k , m , o ) or by two-tailed Student’s t -test ( h ). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The binding affinity of DUSP18 and human GPR54 CT was measured at 40 nM, RUmax = 68.22, Chi = 1.01 ( g ), DUSP18 and mouse Gpr54 336–342 was measured at 1.8 μM, RUmax = 154.6, Chi = 3.42 ( h ). i – l IB and quantification of protein levels analysis of total samples and GST pull-downs using GST proteins including GST, GST-GPR54 CT, GST-GPR54 CT m1 (R336A&P339A), GST-GPR54 CT m2 (delta 339–344) incubated with WCL of 293 T cells transfected with DUSP18-HA ( i ) and quantification of protein levels ( j ), Total samples and GST pull-downs using His-SRC protein (0 or 0.1 μg) purified from Sf9 cells, His-DUSP18 (0, 0.1 or 0.2 μg), and GST proteins purified from E. coli ( k ) and quantification of protein levels ( l ).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Derivative Assay, Transfection, Construct, Purification, Staining, Microscopy, Cell Culture, In Vitro, Incubation, Activity Assay, Confocal Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Kisspeptin-10 binding to Gpr54 in osteoclasts prevents bone loss by activating Dusp18-mediated dephosphorylation of Src

doi: 10.1038/s41467-024-44852-9

Figure Lengend Snippet: a , b IB analysis of lysates from RAW264.7 with indicated dose of Kp-10 treatment for 1 h ( a ) and quantification results ( b ). c Quantitative PCR of Dusp18 mRNA in RAW264.7 cells treated with inhibitors of PLC (U73122, 10 µM), PKC (Staurosporine, 0.25 µM), ERK (LY3214996, 20 µM), and P38 (SB203580, 10 µM) for 1 h, and then incubated with indicated dose of Kp-10 for 30 min. d – i Anti-Flag IP of lysates derived from 293 T cells transfected with indicated constructs and treated with PLC inhibitor (U73122, 10 µM), PKC inhibitor (Staurosporine, 0.25 µM), ERK inhibitor (LY3214996, 10 µM), Ca2+/CaMKII inhibitor (KN-93, 10 µM), and Src inhibitor (Saracatinib, 10 µM) for 1 h and then stimulated with 10 nM Kp-10 for 20 min. Anti-Flag IP of lysates derived from 293 T cells transfected with GPR54-flag and Src-HA constructs ( d ) and quantification results ( e ). Anti-Flag IP of lysates derived from 293 T cells transfected with GPR54-flag and DUSP18-HA constructs ( f ) and quantification results ( g ). Anti-Flag IP of lysates derived from 293 T cells transfected with Src-HA, and DUSP18-flag constructs ( h ) and quantification results ( i ). j , k IB analysis of lysates derived from WT BMMs and Gq/11 KO BMMs with or without Kp-10 treatment for 1 h ( j ) and quantification of results ( k ). l Working model of Kp-10 /Gpr54 mediated Src dephosphorylation. Low dose of Kp-10 (0.1, 1 nM) induced the expression of Dusp18 obviously but not by 10 nM Kp-10, which is dependent on Gq/11 signaling. However, both active Src and DUSP18 were recruited by GPR54 through the PR motif in GPR54 CT along with the increase of Kp-10 dose (1, 10 nM). Therefore, phosphorylation of Src was dose dependently suppressed when GPR54 was activated by Kp-10. Data represent means ± SEM. P values were determined by one-way ANOVA analysis ( b , c , e , g , i , k ). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The binding affinity of DUSP18 and human GPR54 CT was measured at 40 nM, RUmax = 68.22, Chi = 1.01 ( g ), DUSP18 and mouse Gpr54 336–342 was measured at 1.8 μM, RUmax = 154.6, Chi = 3.42 ( h ). i – l IB and quantification of protein levels analysis of total samples and GST pull-downs using GST proteins including GST, GST-GPR54 CT, GST-GPR54 CT m1 (R336A&P339A), GST-GPR54 CT m2 (delta 339–344) incubated with WCL of 293 T cells transfected with DUSP18-HA ( i ) and quantification of protein levels ( j ), Total samples and GST pull-downs using His-SRC protein (0 or 0.1 μg) purified from Sf9 cells, His-DUSP18 (0, 0.1 or 0.2 μg), and GST proteins purified from E. coli ( k ) and quantification of protein levels ( l ).

Techniques: Real-time Polymerase Chain Reaction, Incubation, Derivative Assay, Transfection, Construct, De-Phosphorylation Assay, Expressing, Phospho-proteomics

Journal: Nature Communications

Article Title: Kisspeptin-10 binding to Gpr54 in osteoclasts prevents bone loss by activating Dusp18-mediated dephosphorylation of Src

doi: 10.1038/s41467-024-44852-9

Figure Lengend Snippet: a , c , e , g , i , k Representative micro-CT images of femoral trabecular bone and cortical bone. 4-month-old WT and Gpr54 cKO mice ( n = 7 per group including 4 female and 3 male mice) ( a , c ), 4-month-old WT and Kiss1 cKO mice ( n = 6 per group including 3 female and 3 male mice) ( e , g ) and 8-week-old WT and Dusp18 −/− mice with or without treatment of KX2-391 or (DSS)*6-KP-10 ( n = 7 for WT mice including 3 female and 4 male mice, n = 5 for each other group including 2 female and 3 male Dusp18 −/− mice) ( i , k ). Scale bar, 500 µm. b , d , f , h , j , l Bone parameters analysis of femurs from the mice above. 2-month-old WT and Gpr54 cKO mice ( b , d ), 4-month-old WT and Kiss1 cKO mice ( f , h ), 4-month-old and Dusp18 −/− mice ( j , l ). BMD bone mineral density, BV/TV bone volume as a fraction of total bone volume, Tb.Th trabecular thickness, Tb.N trabecular number, Tb.Sp trabecular separation. m , o , q Representative TRAP staining images of femurs from the mice above. 4-month-old and Gpr54 cKO mice ( m ), 4-month-old and Kiss1 cKO mice ( o ) and 4-month-old and Dusp18 −/− mice ( q ). Scale bar, 200 µm. n , p , r Osteoclast parameters analysis of femurs from the mice above. 4-month-old WT and Gpr54 cKO mice ( n ), 4-month-old and Kiss1 cKO mice ( p ), 4-month-old and Dusp18 −/− mice ( r ). Data represent means ± SEM. P values were determined by two-tailed Student’s t -test ( b , d , f , h , n , p ) or one-way ANOVA analysis ( j , l , r ). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The binding affinity of DUSP18 and human GPR54 CT was measured at 40 nM, RUmax = 68.22, Chi = 1.01 ( g ), DUSP18 and mouse Gpr54 336–342 was measured at 1.8 μM, RUmax = 154.6, Chi = 3.42 ( h ). i – l IB and quantification of protein levels analysis of total samples and GST pull-downs using GST proteins including GST, GST-GPR54 CT, GST-GPR54 CT m1 (R336A&P339A), GST-GPR54 CT m2 (delta 339–344) incubated with WCL of 293 T cells transfected with DUSP18-HA ( i ) and quantification of protein levels ( j ), Total samples and GST pull-downs using His-SRC protein (0 or 0.1 μg) purified from Sf9 cells, His-DUSP18 (0, 0.1 or 0.2 μg), and GST proteins purified from E. coli ( k ) and quantification of protein levels ( l ).

Techniques: Micro-CT, Staining, Two Tailed Test