phosphatase inhibitor cocktail iii  (TargetMol)

Journal: Nature Communications

Article Title: Targeting leucine-rich repeat kinase 2 overcomes resistance to oncolytic herpes simplex virus-based therapies in glioblastoma

doi: 10.1038/s41467-026-70132-9

Figure Lengend Snippet: a Anti-myc immunoprecipitates prepared from 293 T cells transfected with the indicated plasmids were subjected to SDS‒PAGE and blotted with the indicated antibodies. The samples derive from the same experiment but different gels for Myc and another for STAT1 were processed in parallel. b Endogenous LRRK2 and STAT1 complexes in U-118 MG cells were isolated by co-immunoprecipitation (co-IP) and detected using anti-LRRK2 antibodies. The samples derive from the same experiment but different gels for LRRK2 and another for STAT1 were processed in parallel. c The interactions between STAT1-Flag and Myc-LRRK2 mutants were detected by a co-IP assay. FL, full-length. The samples derive from the same experiment but different gels for Myc and another for Flag were processed in parallel. d , e The interactions between Myc-LRRK2 and STAT1-HA truncations were detected by a co-IP assay. The samples derive from the same experiment but different gels for Myc and another for HA were processed in parallel. f STAT1-His proteins were incubated with human FL LRRK2 proteins in the presence of ATP. The reaction products were analyzed with the indicated antibodies. CD300A proteins served as a negative control. The samples derive from the same experiment but different gels for Flag and another for His were processed in parallel. g , h 293 T cells were cotransfected with Flag-STAT1 and LRRK2 in the presence or absence of LRRK2-IN-1, and the phosphorylation of STAT1 was analyzed by western blot analysis. The samples derive from the same experiment but different gels for STAT1, another for p-STAT1 701 and another for p-STAT1 727 were processed in parallel. i 293 T cells were cotransfected with Flag-STAT1 and each LRRK2 mutant, either in the presence or absence of LRRK2-IN-1, and STAT1 phosphorylation was assessed via western blot analysis. The samples derive from the same experiment but different gels for STAT1, another for p-STAT1 701 and another for GAPDH were processed in parallel. j Western blotting was performed to assess and quantify phosphorylated STAT1 and HSV-derived GFP in U-87 MG cells treated with fludarabine (10 µM) for 4 h, either in the presence or absence of LRRK2-IN-1, followed by infection with oHSV-GFP (MOI = 1 PFU/cell). The samples derive from the same experiment but different gels for STAT1 and GFP, another for p-STAT1 701 and another for actin were processed in parallel. k Western blotting was performed to assess and quantify phosphorylated STAT1 and HSV-derived GFP in GBM-1 cells. Cells transfected with EV, LRRK2 or LRRK2 D1994A were infected with oHSV-GFP (MOI = 0.5 PFU/cell), with or without LRRK2-IN-1. The samples derive from the same experiment but different gels for STAT1 and GFP, another for p-STAT1 701 and another for actin were processed in parallel. l Western blotting was performed to assess and quantify phosphorylated TBK1, phosphorylated LRRK2, phosphorylated Rab10, and phosphorylated STAT1 in BX795 (10 µM)-treated U-87 cells following infection with oHSV-GFP (MOI = 0.5 PFU/cell). The samples derive from the same experiment but different gels for p-TBK1, another for p-LRRK2, another for LRRK2, another for p-Rab10 and p-STAT1, another for STAT1 and another for actin were processed in parallel. Western blot analyses were independently repeated twice for panels ( a – d and f )and three times for panels ( e , g and i – l ), with similar results. The data are presented as the mean ± SD from three independent experiments ( h – l ), and P values were calculated using an ordinary one-way ANOVA ( h – l ).

Article Snippet: The following chemicals and recombinant proteins were used: Bioactive Compound Library (MedChemExpress, HY-L001), LRRK2-IN-1 (MedChemExpress, HY-10875), DCLK1-IN-1 (MedChemExpress, HY-135985), MLi-2 (MedChemExpress, HY-100411), Fludarabine (MedChemExpress, HY-B0069), PFE-360 (MedChemExpress, HY-120085), BX795 (TopScience, T1830), Phosphatase inhibitor cocktail III (TopScience, C0004), Protease inhibitor cocktail (MedChemExpress, HY-K0011), and RIPA lysis buffer (Beyotime, P0013C).

Techniques: Transfection, Isolation, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Negative Control, Phospho-proteomics, Western Blot, Mutagenesis, Derivative Assay, Infection