Journal: PLoS Pathogens

Article Title: Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

doi: 10.1371/journal.ppat.1005660

Figure Lengend Snippet: A) Coprecipitation of bacterially translocated YopM-SBP-CBP with DDX3, RSK1 and PKN in J774A.1 cells . J774A.1 cells were infected with WA314ΔYopM(pYopM-SBP-CBP), WA314ΔYopM(pYopM) or WA314ΔYopE(pYopE-SBP-CBP). Proteins eluting consecutively from streptavidin-sepharose (biotin elution) and calmodulin-sepharose (boiling in sample buffer) were analyzed by Western blot using indicated antibodies. B) GST-YopM pull-down of endogenous DDX3 . GST-myc-YopM or GST expressed in HEK293T cells were precipitated with glutathione sepharose beads and analyzed by Western blot using indicated antibodies. C) Myc-YopM co-immunoprecipitates with endogenous DDX3 . Endogenous DDX3 was immunoprecipitated in HEK293T cells expressing myc-YopM. Precipitates and whole cell lysates (WCL) were analyzed by Western blot using indicated antibodies. D) Bacterially translocated YopM co-immunoprecipitates with endogenous DDX3 . Endogenous DDX3 was immunoprecipitated in HEK293T cells infected with WA314ΔYopM(pYopM) or WA314ΔYopM for 90 min. Precipitates and whole cell lysates (WCL) were analyzed by Western blot using indicated antibodies. E) DDX3 and PKN mutually exclude each other in RSK1/YopM containing complexes . HEK293T cells expressing either DDX3-FLAG (left panel) or PKN-FLAG (right panel) and where indicated myc-YopM were lysed and subjected to anti-FLAG immunoprecipitation. Precipitates and whole cell lysates (WCL) were analyzed by Western blot using indicated antibodies. In DDX3-FLAG immunoprecipitates, myc-YopM, RSK1 but no PKN and in PKN-FLAG immunoprecipitates myc-YopM, RSK1 but no DDX3 were detected.

Article Snippet: The following expression plasmids have been described previously: HA-tagged RSK1 (Addgene plasmid 13841) [ ] and FLAG-tagged PKN1 [ ].

Techniques: Infection, Western Blot, Immunoprecipitation, Expressing

Journal: PLoS Pathogens

Article Title: Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

doi: 10.1371/journal.ppat.1005660

Figure Lengend Snippet: A) Coexpression of HA-RSK1 and myc-YopM increases phosphorylation of nuclear RSK1 . (Left panel) HEK293T cells were transfected with indicated expression vectors and cytosolic- (C) and nuclear (N) fractions were investigated by immunoblot using indicated antibodies. Anti-pS221- and Anti-pS380 antibodies detect the respective phosphorylated amino acids in RSK1. Exposure time of the anti-RSK1 panel in the HA-RSK1 expressing cells (right, WB 1) is about 1/5 of that of the respective panel in control cells (left). (Right panel) Intensities of the pS221- pS380- and RSK1 protein bands from WB 1 and WB 2, the latter derived from a second identical experiment were quantified and intensity ratios of pS221/RSK1 and pS380/RSK1 were calculated. B) Leptomycin B treatment and DDX3 knockdown increase phosphorylation of nuclear RSK1 . (Left panel) HEK293T cells were not treated (Ctrl) or treated with 25 nM LMB for 4 h. (middle and right panels) HEK293T cells were treated with control siRNA (siCtrl) or DDX3 siRNA (siRNA No. 3) for 24 h. Cells were infected with Yersinia WA-C(pTTSS+YopM) (middle panel) or WA-C(pTTSS) (right panel) for 90 min and cytosolic- (C) and nuclear (N) fractions were investigated by immunoblot using indicated antibodies. Bottom panel shows percentage of total RSK1 present in the C- and N fraction of cells in each condition. C) Leptomycin B treatment and DDX3 knockdown increases nuclear YopM . HEK293T cells were not treated or treated with 25 nM LMB for 4 h or DDX3 siRNA (No. 3) for 24 h, infected with Yersinia WA-C(pTTSS+YopM) for 90 min and nuclear fractions of cells were investigated by anti-YopM immunoblot. Band intensities of YopM signals were determined by ImageJ. Each bar represents mean ± SEM of values from 4 experiments for LMB treatment and 3 experiments for DDX3 siRNA treatment; *p<0.05 (Bonferroni's multiple comparisons test). D) YopM_1–481 mutant defective in interaction with RSK accumulates in the nucleus and is exported from the nucleus in a LMB- and DDX3 dependent fashion . HEK293T cells were not treated (Ctrl), treated with 25 nM LMB for 4 h or DDX3 siRNA (siRNA No. 3) for 24 h and transfected with indicated expression vectors. Cytosolic- (C) and nuclear (N) fractions of cells in each condition were investigated by immunoblot using indicated antibodies. Bottom panel shows percentage of total myc-YopM present in the C- and N fraction of cells in the respective condition. See also for immunoprecipitaton of myc-YopM; myc-YopM_1–481 and myc-YopM_34–481 to verify the incapability of YopM lacking the C-terminus to bind RSK. E) YopM_1–481 mutant is unable to stimulate phosphorylation of RSK . Indicated myc-YopM constructs or empty vector were expressed in HEK293T cells and cytosolic (C) or nuclear (N) fractions were analyzed by Western blot using indicated antibodies.

Article Snippet: The following expression plasmids have been described previously: HA-tagged RSK1 (Addgene plasmid 13841) [ ] and FLAG-tagged PKN1 [ ].

Techniques: Transfection, Expressing, Western Blot, Derivative Assay, Infection, Mutagenesis, Construct, Plasmid Preparation

Journal: Pharmaceuticals

Article Title: BMP7 Increases UCP1-Dependent and Independent Thermogenesis with a Unique Gene Expression Program in Human Neck Area Derived Adipocytes

doi: 10.3390/ph14111078

Figure Lengend Snippet: BMP7 treatment upregulated creatine futile cycle-mediated thermogenesis in SC and DN derived differentiated adipocytes. SC and DN preadipocytes were differentiated and treated as in , , , and . ( A ) Representative time lapse OCR curve after indicated treatments, followed by quantification of creatine cycle related (β-GPA inhibited) OCR ( n = 5). ( B ) Quantification of gene expression of CKMT2 by RNA-sequencing ( n = 9) and RT-qPCR ( n = 5), followed by protein expression ( n = 5). ( C ) Quantification of gene expression of CKB by RNA-sequencing ( n = 9), followed by protein expression ( n = 4). ( D ) Quantification of gene expression of TNAP by RNA-sequencing ( n = 9). Data expressed as mean ± SD. RT-qPCR was normalized to GAPDH, protein expression was normalized to β-actin. * p < 0.05, ** p < 0.01. Statistics: one-way ANOVA with Tukey’s post-test.

Article Snippet: The following primary antibodies were used overnight in 1% skimmed milk solution: anti-UCP1 (1:750, R&D Systems, MAB6158), anti-pCREB (1:1000, Merck-Millipore, Burlington, MA, USA, 05-667), anti-CREB (1:1000, Abcam, Cambridge, UK, ab31387), anti-PGC1α (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, H-300), anti-CKMT2 (1:1000, Novus Biologicals, Centennial, CO, USA, NBP2-13841), anti-CKB (1:1000, Novus Biologicals, NBP1-84460), anti-CRYAB (1:1000, Novus Biologicals, NB100-2519), anti-β actin (1:5000, Novus Biologicals, A2066), anti-OXPHOS (1:1000, Abcam, ab110411), anti-Aggrecan (1:1000, Novus Biologicals, NB100-74350), and anti-ID1 (1:1000, Novus Biologicals, JM92-13).

Techniques: Derivative Assay, Expressing, RNA Sequencing Assay, Quantitative RT-PCR

Journal: iScience

Article Title: RNPS1 in PSAP complex controls periodic pre-mRNA splicing over the cell cycle

doi: 10.1016/j.isci.2024.111400

Figure Lengend Snippet:

Article Snippet: Rabbit anti-SAP18 , Proteintech , #13841-1-AP.

Techniques: Virus, Recombinant, Transfection, Software