Journal: PLoS ONE

Article Title: Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

doi: 10.1371/journal.pone.0125387

Figure Lengend Snippet: Cells were fixed, TRAP stained, and visualized by light microscopy. (A) Representative 100x images of female (top) and male (bottom) mouse bone marrow cells defined by CD11b surface expression driven to form osteoclasts (OCs) with M-CSF (25ng/mL) and RANKL (50 ng/mL). Female OCs (black arrow) were magnified 3x and inset (top). TRAP positive cells with three or more nuclei (OCs) from (B) female and (C) male mice were enumerated from three random images at days 3 (top) and 5 (bottom) of RANKL treatment. Osteoclast size from female (B, right) and male (C, right) was measured by pixels per osteoclast using Adobe PhotoShop CS. Data are expressed as means ± SE compared to Mk2 +/+ controls (* P ≤0.05, ** P ≤0.01, *** P ≤0.001).

Article Snippet: Membranes were incubated in Cell Signaling primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and GAPDH (14C10, rabbit mAb) at a 1:1000 dilution in 5% BSA in TBS-T overnight at 4°C.

Techniques: Staining, Light Microscopy, Expressing

Journal: PLoS ONE

Article Title: Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

doi: 10.1371/journal.pone.0125387

Figure Lengend Snippet: (A) Representative flow cytometry of bone marrow from male and female Mk2 +/+ and Mk2 -/- mice. (B) Enumeration of Gr-1 - CD11b lo cells as a percent of the hematopoietic stem cell population (HSC, top) and CD115+ as a percent of Gr-1 - CD11b lo cells (bottom). (C) Enumeration of CD11b surface expression in female (left) and male (right) mouse bone marrow cells. Data are expressed as means ± SE compared to Mk2 +/+ controls (* P ≤0.05).

Article Snippet: Membranes were incubated in Cell Signaling primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and GAPDH (14C10, rabbit mAb) at a 1:1000 dilution in 5% BSA in TBS-T overnight at 4°C.

Techniques: Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

doi: 10.1371/journal.pone.0125387

Figure Lengend Snippet: (A) Representative western blot of pre-osteoclasts from male mice primed with M-CSF (10 ng/mL) for two days and stimulated with RANKL (100 ng/mL) for the indicated minutes (min). Densiometric analysis of (B) p38 (left) and (C) p-p38 (right) as a percent of GAPDH. Data are expressed as means ± SE compared to Mk2 +/+ controls (* P ≤0.05).

Article Snippet: Membranes were incubated in Cell Signaling primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and GAPDH (14C10, rabbit mAb) at a 1:1000 dilution in 5% BSA in TBS-T overnight at 4°C.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

doi: 10.1371/journal.pone.0125387

Figure Lengend Snippet: (A) Representative confocal images of male dOCP lo cells treated with M-CSF (control) or M-CSF and RANKL for 30 minutes. A 3x magnified portion of p-p38 was inset (middle,left). (B) Pearson’s r correlation coefficients for NFATc1 and DAPI colocalization. (C) Pearson’s r correlation coefficients for p-p38 and DAPI colocalization. (D) Pearson’s r correlation coefficients for NFATc1 and p-p38 colocalization. Data are expressed as means ± SE compared to Mk2 +/+ controls (* P ≤0.05).

Article Snippet: Membranes were incubated in Cell Signaling primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and GAPDH (14C10, rabbit mAb) at a 1:1000 dilution in 5% BSA in TBS-T overnight at 4°C.

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Post-Transcriptional Inflammatory Response to Intracellular Bacterial c-di-AMP

doi: 10.3389/fimmu.2019.03050

Figure Lengend Snippet: P38 inhibition leads to the reduction in the expression of ARE-containing mRNAs and induction of TTP. (A) Upper panel multiplex ELISA for the indicated cytokines. BMDM were treated with 5 μM SB203580 or DMSO during the treatment with c-di-AMP for 4 h. Cell medium was used for multiplex ELISA. Lower panel the BMDM were lysed, total RNA was prepared, and the mRNA levels of the indicated cytokines were determined. Individual values of at least three independent experiments are represented as fold of levels in DMSO control treated cells. Means ± SEMs are shown. Asterisk represents P <0.05 of paired t -tests. (B) Similar experiment like (A) but with RAW264.7 cells. (C) RAW264.7 cells were treated with PS alone or PS in the presence of c-di-AMP for the indicated time points. The cells were lysed in SDS loading buffer, and western blots were performed and developed with an antibody specific for phosphorylated p38. β-Actin was used as loading control. (D) Western blot similar to (C) with one PS control at 6 h and at time points as indicated. The blot was developed with anti-TTP antibody and β-actin. (E) RAW264.7 cells were treated for the indicated time points with PS+c-di-AMP or LPS. The cells were lysed in SDS loading buffer, and western blots were performed and developed with antibodies specific for TTP, phosphorylated p38 (P-p38), p38, phosphorylated MK2 (P-MK2), MK2, and β-actin.

Article Snippet: P38 antibody was purchased from Santa Cruz Biotechnology; anti-phospho-p38 (Thr180/Tyr182) was purchased from Millipore/Merck; β-actin, MK2, and P-MK2 (Thr334) antibodies were purchased from Cell Signaling.

Techniques: Inhibition, Expressing, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Control, Western Blot

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 ) Expression of Rab1A, P-S6K, S6K, P-AKT, AKT, P-ERK, ERK, P-MK2, MK2, P-c-Jun and c-Jun was examined in a panel of lung cancer cell lines by immunoblot. GAPDH served as a loading control. ( B ) Quantitative analysis of Rab1A protein levels in cell lines. The densitometry results for each Rab1A band were normalized to GAPDH, and the ratios between cancer cell lines and BEAS-2B were calculated and results represented in a histogram. Data represent means ± SD of three independent experiments. ( C-F ) Correlation plots of the expression level of Rab1A and P-S6K1 (T389; C ), P-AKT (S473; D ), P-ERK (T202/Y204; E ), P-MK2 (T334; F ) and P-c-Jun (S63; G ). Shown are arbitrary units.

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: Expressing, Western Blot, Control

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: 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