MDA-MB-231 cells were treated with 100 g/mL cycloheximide in the presence of DMSO, 10 nM MG132, or 0.5 M of ibrutinib for the indicated times. KO cells relative to parental controls. (F) SRMS(K258A) is enzymatically inactive. Myc-SRMS(WT) and Myc-SRMS(K258A) were expressed in SRMS KO U2OS cells. Cell lysates were immunoprecipitated with anti-Myc antibody and blotted with anti-p-Tyr antibody to detect Tianeptine SRMS autophosphorylation. (G) Myc-SRMS(WT) and Myc-SRMS(K258A) were transiently expressed in U2OS cells stably expressing GFP-LC3. Cells were fixed and stained with anti-Myc antibody to detect transfected cells. Representative images are shown. For quantitation, see Fig 1E. (H) MS/MS fragmentation data for human Tianeptine SRMS AA 374C387 sequence LLKDDIY(+79.97)SPSSSK, M/z 810.3703, z2, showing b/y ions. MS/MS fragment ions at M/z 941.42 (b7) and M/z 922.34 (y8) represent characteristic ions that unambiguously identify Y380 phosphorylation. The data underlying the figure can be found in S1 Data. IP, immunoprecipitation; KO, knockout; MEF, mouse embryonic fibroblast; MS/MS, tandem mass Tianeptine spectrometry; RNAi, RNA interference; SRMS, Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites; WT, wild-type.(PDF) pbio.3001281.s001.pdf (4.1M) GUID:?8C21A3B7-8882-47B8-8298-1BD38DDF189E S2 Fig: Ibrutinib blocks SRMS kinase activity and increases autophagy. (A) SRMS overexpression increases p-Tyr immunoreactivity. HeLa cells were transiently transfected with the indicated constructs. Twenty-four hours later, lysates were collected and analyzed by western blot using the indicated antibodies. (B) Ibrutinib inhibits SRMS activity in a dose-dependent manner. HEK293 cells stably expressing Myc-SRMS(WT) were treated with the indicated compounds at the indicated concentrations for 2 hours. Cell lysates were Rabbit Polyclonal to Cytochrome P450 21 subjected to immunoblotting with indicated antibodies. (C) Ibrutinib activates LC3 lipidation in a dose-dependent manner. Parental MDA-MB-231 cells were treated with ibrutinib at the indicated concentrations for 4 hours. Cell lysates were immunoblotted with anti-LC3 antibody. (D) Ibrutinib activates autophagosome formation in a dose-dependent manner. U2OS cells stably expressing GFP-LC3 were treated with ibrutinib at the indicated concentrations for 4 hours. GFP-LC3 puncta were detected by confocal microscopy. Representative images are shown. (ECG) Ibrutinib activates autophagy in an SRMS-dependent manner as measured by acridine orange. Parental or SRMS KO U2OS cells were treated with DMSO or ibrutinib (0.5 M or as indicated) for 8 hours. Cells were after that stained with 1 g/mL acridine orange for 20 a few minutes and imaged on the indicated wavelengths. Representative pictures are proven (E) along with quantitation (F, G). For -panel F, 10 pictures (123 cells) for parental and = 8 pictures (130 cells) for SRMS KO. G displays mean +/? regular deviation of = 10 pictures (123 cells), = 11 pictures (131 cells), = 8 pictures (100 cells), and = 9 pictures (79 cells) for parental and = 8 pictures (130 cells), = 8 pictures (139 cells), = 8 pictures (128 cells), and = 9 pictures (181 cells) for SRMS KO (still left to correct). * 0.05, ** 0.01, *** 0.001, **** 0.0001, check. (H) Ibrutinib induces autophagosome biogenesis and autophagosomeClysosome fusion. U2Operating-system cells stably expressing RFP-GFP-LC3 had been treated with 1 M ibrutinib for 12 hours. Cells had been imaged by IncuCyte. Cells having a lot more than 3 puncta had been counted. Mean + SD of = 15 cells per condition is normally proven. ** 0.01, *** 0.001, **** 0.0001, check. (I) Ibrutinib induces autophagic flux, but acalabrutinib will not. U2Operating-system Autophagy LC3 HiBiT Reporter cells had been plated at Tianeptine 8,000 cells per well. (J) SRMS kinase activity Tianeptine restrains LC3 lipidation. Myc-SRMS(WT) and Myc-SRMS(T302M) had been portrayed in SRMS KO U2OS cells and treated with 0.5 M ibrutinib for 4 hours. Cell lysates had been immunoprecipitated with anti-Myc antibody and blotted with indicated antibodies. The info underlying the amount are available in S1 Data. IP, immunoprecipitation; KO, knockout; SRMS, Src-related kinase missing C-terminal regulatory tyrosine and N-terminal myristylation sites; WT, wild-type.(TIF) pbio.3001281.s002.tif (5.5M) GUID:?E4A0E5CF-4FA7-4D62-BD7F-A1BC4DA77478 S3 Fig: SRMS interacts with FKBP51 through its kinase domain. (A, B) SRMS interacts with FKBP51 through its kinase domains. SRMS truncated constructs (schematized within a) had been transfected with Flag-FKBP51 in HEK293FT cells. Cell lysates had been.