Journal: Infection and Immunity

Article Title: Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss

doi: 10.1128/iai.00552-16

Figure Lengend Snippet: Figure 5B). These results suggest that MK2 differentially regulates chemokines in the 407

Article Snippet: Membranes 204 on N ovem ber 1, 2016 by R Y E R S O N U N IV http://iai.asm .org/ D ow nloaded from 10 were incubated in the following primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), MK2 205 (rabbit polyclonal), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and 206 GAPDH (14C10, rabbit mAb) (Cell Signaling Technology Inc., Beverly, MA, USA) at a 1:1000 207 dilution overnight at 4 °C.

Techniques:

Journal: Infection and Immunity

Article Title: Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss

doi: 10.1128/iai.00552-16

Figure Lengend Snippet: Figure 6D). Furthermore, we elucidated that MK2 signaling did not alter the percent of 421

Article Snippet: Membranes 204 on N ovem ber 1, 2016 by R Y E R S O N U N IV http://iai.asm .org/ D ow nloaded from 10 were incubated in the following primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), MK2 205 (rabbit polyclonal), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and 206 GAPDH (14C10, rabbit mAb) (Cell Signaling Technology Inc., Beverly, MA, USA) at a 1:1000 207 dilution overnight at 4 °C.

Techniques:

Journal: Infection and Immunity

Article Title: Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss

doi: 10.1128/iai.00552-16

Figure Lengend Snippet: Figure 3: MK2 regulates chemokines in A. actinomycetemcomitans challenged macrophages 672

Article Snippet: Membranes 204 on N ovem ber 1, 2016 by R Y E R S O N U N IV http://iai.asm .org/ D ow nloaded from 10 were incubated in the following primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), MK2 205 (rabbit polyclonal), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and 206 GAPDH (14C10, rabbit mAb) (Cell Signaling Technology Inc., Beverly, MA, USA) at a 1:1000 207 dilution overnight at 4 °C.

Techniques:

Journal: Infection and Immunity

Article Title: Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss

doi: 10.1128/iai.00552-16

Figure Lengend Snippet: Figure 5: MK2 differentially regulates chemokines CCL3 and CCL4. A) ELISA 690

Article Snippet: Membranes 204 on N ovem ber 1, 2016 by R Y E R S O N U N IV http://iai.asm .org/ D ow nloaded from 10 were incubated in the following primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), MK2 205 (rabbit polyclonal), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and 206 GAPDH (14C10, rabbit mAb) (Cell Signaling Technology Inc., Beverly, MA, USA) at a 1:1000 207 dilution overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Expression of Rab1A is upregulated in human lung cancer and associated with tumor size and T stage

doi: 10.18632/aging.101087

Figure Lengend Snippet: ( A ) The effects of Rab1A knockdown was examined in H838, H1975, H650 and A549 cells by immunoblot. Levels of P-S6K1 (T389), S6K, P-AKT (S473) and AKT are shown. β-tubulin was used as a loading control. ( B ) Rab1A was knocked down in H838, H1975, H650 and A549 cells. The relative growth of these cells was analyzed using an MTT assay. Data represent means ± SD of three independent triplicate experiments. si-NC, negative control siRNA; si-Rab1A, Rab1A-specific siRNA.

Article Snippet: Reagents were obtained from the following sources: antibody for Rab1A, Proteintech Group (Rosemont, IL); antibodies for P-S6K1(T398), S6K1, P-AKT(S473), AKT, P-ERK(T202/Y204), ERK, P-MK2(T334), MK2 and Tubulin, Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Western Blot, Control, MTT Assay, Negative Control

Journal: The EMBO Journal

Article Title: A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis

doi: 10.1038/emboj.2013.223

Figure Lengend Snippet: Rapid displacement of AZI1, PCM1, and CEP290 from centriolar satellites in response to UV radiation. (A) U2OS cells were mock treated or exposed to UV or IR, fixed 1 h later and co-immunostained with AZI1 and γ-tubulin antibodies. Scale bar, 10 μm. (B) U2OS cells subjected to UV or not and treated as in (A) were co-immunostained with PCM1 and γ-tubulin antibodies. Scale bar, 10 μm. (C) As in (B), except that cells were co-immunostained with CEP290 and γ-tubulin antibodies. Scale bar, 10 μm. (D) U2OS cells were exposed or not to UV, collected 1 h later and separated into subcellular fractions. Cytoplasm- and cytoskeleton-enriched fractions were immunoblotted with indicated antibodies. (E) U2OS cells were left untreated, transfected with siRNAs against AZI1 or PCM1, or exposed to UV and subsequently prepared for electron microscopy combined with PCM1 immunogold labelling (black dots). Centrioles (rod shaped) are indicated by arrows. Scale bars, 1000, nm. (F) As in (B), except that cells were co-immunostained with OFD1 and γ-tubulin antibodies. Scale bar, 10 μm. High-magnification images are shown in Supplementary Figure S2. (G) Quantification of centriolar satellite localization of PCM1 and OFD1, analysed as in (B) and (F). At least 100 cells per condition were counted. Results depict the mean (±s.d.) of three independent experiments.

Article Snippet: Antibodies used in this study included: rabbit polyclonals to AZI1 (A301-415A, Bethyl; ab84864, Abcam), PCM1 (A301-150A, Bethyl), MIB1 (NBP1-95846, Novus), p38 (9212S, Cell Signaling), MK2 (3042S, Cell Signaling), CEP290 (ab84870, Abcam), NF-κB-p65 (Cell Signaling), and Histone H3-pSer10 (06-570, Millipore); mouse monoclonals to p-p38 (T180/Y182) (9216S, Cell Signaling), p-MK2 (T334) (3007S, Cell Signaling), GFP (sc-9996, Santa Cruz), γ-tubulin (T5326, Sigma) and Acetylated tubulin (ab24610, Abcam); rat monoclonal to HA tag (11867423991, Roche).

Techniques: Transfection, Electron Microscopy

Journal: The EMBO Journal

Article Title: A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis

doi: 10.1038/emboj.2013.223

Figure Lengend Snippet: The stress-responsive kinase p38 is required for dispersal of centriolar satellite factors in response to a range of cell stresses. (A) U2OS cells were exposed to UV, heat shock, or the transcriptional inhibitors DRB or Actinomycin D, fixed and immunostained with AZI1 antibody. Scale bar, 10 μm. (B) U2OS cells were incubated with p38 inhibitor (p38i) for 1 h or transfected with p38 siRNA for 72 h. Subsequently, cells were irradiated or not with UV, fixed 1 h later and stained with AZI1 antibody. Scale bar, 10 μm. (C) U2OS cells treated as in (B) were stained with AZI1 and γ-tubulin antibodies. Scale bar, 10 μm. (D) U2OS cells treated as in (B) were immunoblotted with indicated antibodies. (E) Cytoskeletal and cytoplasmic fractions of cells treated with p38 inhibitor and/or UV for 1 h prior to harvest were analysed with the indicated antibodies. (F) U2OS cells transfected with WT or kinase-dead (K82A) versions of FLAG-tagged MKK6 were fixed and immunostained with AZI1 and FLAG antibodies. Scale bars, 10 μm.

Article Snippet: Antibodies used in this study included: rabbit polyclonals to AZI1 (A301-415A, Bethyl; ab84864, Abcam), PCM1 (A301-150A, Bethyl), MIB1 (NBP1-95846, Novus), p38 (9212S, Cell Signaling), MK2 (3042S, Cell Signaling), CEP290 (ab84870, Abcam), NF-κB-p65 (Cell Signaling), and Histone H3-pSer10 (06-570, Millipore); mouse monoclonals to p-p38 (T180/Y182) (9216S, Cell Signaling), p-MK2 (T334) (3007S, Cell Signaling), GFP (sc-9996, Santa Cruz), γ-tubulin (T5326, Sigma) and Acetylated tubulin (ab24610, Abcam); rat monoclonal to HA tag (11867423991, Roche).

Techniques: Incubation, Transfection, Irradiation, Staining

Journal: The EMBO Journal

Article Title: A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis

doi: 10.1038/emboj.2013.223

Figure Lengend Snippet: MIB1 ubiquitin ligase activity suppresses primary cilium formation. (A) Human RPE1 cells were serum starved by culturing in medium containing 0.25% FCS, fixed and immunostained with antibodies against AZI1 and acetylated tubulin (Ac-tubulin). Scale bar, 10 μm. (B) Effect of AZI1 or PCM1 knockdown on primary cilium formation in cells treated as in (A) was analysed by immunostaining with antibodies against pericentrin and acetylated tubulin (left). Data from three independent experiments were quantified (right). At least 300 cells were counted in each experiment. Scale bar, 10 μm. (C) RPE1 cells were transfected with GFP-MIB1 WT or CI for 24 h and serum starved for 16 h. Cells were then fixed, immunostained with antibody to acetylated tubulin, and the fraction of GFP-positive cells with a cilium was determined. The experiment was performed in triplicates and at least 150 GFP-positive cells were counted for each condition. (D) RPE1 cells were transfected with control (CTRL) or MIB1 siRNAs for 72 h and cultured in normal medium containing 10% serum (10% FCS). Spontaneous cilium formation was visualized and quantified as in (B). Scale bar, 10 μm. Data in (B–D) were analysed by pairwise two-tailed t-test. **P<0.01, ***P<0.001. (E) GFP-MIB1 autoubiquitylation was analysed by transfecting RPE1 cells with GFP-MIB1 and S-Myc-ubiquitin plasmids. Cells were then grown in medium containing high (10% FCS) or low (0.25% FCS) serum concentrations for 24 h and collected. Whole-cell extracts (WCEs) were subjected to pull-down with S-tag agarose and immunoblotted with GFP antibody.

Article Snippet: Antibodies used in this study included: rabbit polyclonals to AZI1 (A301-415A, Bethyl; ab84864, Abcam), PCM1 (A301-150A, Bethyl), MIB1 (NBP1-95846, Novus), p38 (9212S, Cell Signaling), MK2 (3042S, Cell Signaling), CEP290 (ab84870, Abcam), NF-κB-p65 (Cell Signaling), and Histone H3-pSer10 (06-570, Millipore); mouse monoclonals to p-p38 (T180/Y182) (9216S, Cell Signaling), p-MK2 (T334) (3007S, Cell Signaling), GFP (sc-9996, Santa Cruz), γ-tubulin (T5326, Sigma) and Acetylated tubulin (ab24610, Abcam); rat monoclonal to HA tag (11867423991, Roche).

Techniques: Ubiquitin Proteomics, Activity Assay, Knockdown, Immunostaining, Transfection, Control, Cell Culture, Two Tailed Test

Journal: The EMBO Journal

Article Title: A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis

doi: 10.1038/emboj.2013.223

Figure Lengend Snippet: Cell stresses induce p38-dependent primary cilium formation in proliferating cells. (A) RPE1 cells were exposed or not to UV (10 J/m2), kept at normal serum concentrations (10% FCS), and fixed 18 h later. Spontaneous cilium formation was analysed by immunostaining with antibodies against pericentrin and acetylated tubulin (left). Data from three independent experiments were quantified (right). At least 300 cells were counted in each experiment. Scale bars, 10 μm. (B) As in (A), except that cells were mock treated or subjected to heat shock for 30 min and fixed 18 h later. (C) Exponentially growing RPE1 cells were exposed to UV (10 J/m2) or subjected to heat shock. Where indicated, p38 inhibitor (p38i) was added to the cultures 30 min before treatment. After an additional 16 h, cells were fixed, co-immunostained with antibodies to acetylated tubulin and pericentrin, and the proportion of cilium-positive cells was determined. The experiment was performed in triplicates and at least 300 cells were counted for each condition. (D) RPE1 cells were transfected with control (−) or MIB1 siRNAs for 48 h, exposed to UV (10 J/m2) and fixed 16 h later. Cells were then immunostained and analysed as in (C). Data in (A–D) were analysed by pairwise two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001. (E) Model for stress-induced reorganization of centriolar satellites and ciliogenesis. During unperturbed cell proliferation, AZI1, PCM1, and CEP290 are associated with centriolar satellites and ubiquitylated by the MIB1 E3 ubiquitin ligase, which is itself a component of centriolar satellites. In response to cell stresses such as UV radiation, heat shock, and transcription blocks, activation of the p38 kinase triggers acute dissociation of these proteins from centriolar satellites, leading to enhanced AZI1–PCM1 interaction. In parallel, cell stress also causes p38-independent inactivation of MIB1 and concomitant loss of AZI1, PCM1, and CEP290 ubiquitylation. Cooperatively, these stress-induced pathways cooperate to relieve inhibitory constraints to primary cilium formation in proliferating cells.

Article Snippet: Antibodies used in this study included: rabbit polyclonals to AZI1 (A301-415A, Bethyl; ab84864, Abcam), PCM1 (A301-150A, Bethyl), MIB1 (NBP1-95846, Novus), p38 (9212S, Cell Signaling), MK2 (3042S, Cell Signaling), CEP290 (ab84870, Abcam), NF-κB-p65 (Cell Signaling), and Histone H3-pSer10 (06-570, Millipore); mouse monoclonals to p-p38 (T180/Y182) (9216S, Cell Signaling), p-MK2 (T334) (3007S, Cell Signaling), GFP (sc-9996, Santa Cruz), γ-tubulin (T5326, Sigma) and Acetylated tubulin (ab24610, Abcam); rat monoclonal to HA tag (11867423991, Roche).

Techniques: Immunostaining, Transfection, Control, Two Tailed Test, Ubiquitin Proteomics, Activation Assay