Journal: PLoS Genetics

Article Title: Regulation of Caenorhabditis elegans p53/CEP-1–Dependent Germ Cell Apoptosis by Ras/MAPK Signaling

doi: 10.1371/journal.pgen.1002238

Figure Lengend Snippet: (A) Phylogenic tree showing the evolutionary relationships of human (Hs), mouse (Mm), Drosophila (Dmel), C. elegans (Ce) and C. briggsae (Cb) MAPK phosphatases. LIP-1 is highlighted by the green oval within the DUSP6/7/9 cluster (blue oval). The other C. elegans MAPK phosphatase VHP-1 is highlighted by the grey oval. (B) In vivo dephosphorylation assays. Increasing amounts of Myc-tagged LIP-1 was expressed in Cos-1 cells and the phosphorylation of endogenous ERKs 1 and 2 (pERK), JNK (pJNK), or p38 (pp38) in response to either serum stimulation (ERK) or anisomycin (JNK and p38) was monitored by western blotting. Myc-tagged human DUSP5 and DUSP1 were used as positive controls to inactivate ERK and JNK/p38, respectively. (C) Sequence alignment of human DUSP6, DUSP7, DUSP9, and LIP-1, showing the conserved Kinase Interaction Motif (KIM). Increasing amounts of either Myc-tagged wild type or a mutant in which the conserved arginine residues (indicated by asterisks) of the KIM were mutated to alanine were expressed in Cos-1 cells and the phosphorylation of ERK1/2 was assessed. Tagged human DUSP1 was used as a positive control.

Article Snippet: Western blot analysis was performed using ERK (K-23, Santa Cruz 1∶2000, rabbit) and phosphorylated ERK (clone MAPK-YT, Sigma, 1∶2000, mouse) specific antibodies that cross react with MPK-1 and phosphorylated MPK-1 , , or anti-GLD-1 (1∶500, rabbit, this study) and HRP conjugated secondary antibodies (anti-rabbit-HRP and anti-mouse-HRP, DakoCytomation 1∶2000).

Techniques: In Vivo, De-Phosphorylation Assay, Western Blot, Sequencing, Mutagenesis, Positive Control

Journal: PLoS Genetics

Article Title: Regulation of Caenorhabditis elegans p53/CEP-1–Dependent Germ Cell Apoptosis by Ras/MAPK Signaling

doi: 10.1371/journal.pgen.1002238

Figure Lengend Snippet: (A and B) Wild type and let-60(ga89) and (C) wild type and let-60(n1046) worms were irradiated and allowed to recover at 20°C, and apoptotic corpses were scored after the stated time points. The scoring of apoptotic corpses was performed as in . (D) Activated (phosphorylated) MPK-1 levels differ between let-60(ga89) and let-60(n1046) worms. Protein was extracted from young adult worms (24 hours post L4 larval stage) and equal amounts were loaded onto SDS-PAGE gels. Activated MPK-1 was detected by an anti-P-ERK antibody, total MPK-1 by an anti-ERK antibody, and α-tubulin was used to control for loading. The ratio of phosphorylated MPK-1 to total MPK-1 (Ratio P∶T, normalised against wild type) is shown for the two isoforms.

Article Snippet: Western blot analysis was performed using ERK (K-23, Santa Cruz 1∶2000, rabbit) and phosphorylated ERK (clone MAPK-YT, Sigma, 1∶2000, mouse) specific antibodies that cross react with MPK-1 and phosphorylated MPK-1 , , or anti-GLD-1 (1∶500, rabbit, this study) and HRP conjugated secondary antibodies (anti-rabbit-HRP and anti-mouse-HRP, DakoCytomation 1∶2000).

Techniques: Irradiation, SDS Page

Journal: PLoS Genetics

Article Title: Regulation of Caenorhabditis elegans p53/CEP-1–Dependent Germ Cell Apoptosis by Ras/MAPK Signaling

doi: 10.1371/journal.pgen.1002238

Figure Lengend Snippet: (A and D) Wild type, (B and E) lip-1(gt448) , and (C and F) lip-1(zh15) worms (24 hours post L4 larval stage) were treated with 0 or 60 Gy of gamma irradiation and allowed to recover for two hours. Germlines were then dissected and processed for immunofluorescence using an anti-P-MPK-1 antibody (red). Nuclei were stained with DAPI (blue). All images show the germlines from the mid pachytene to the diakenesis region, the loop region (in which the cells are in late pachytene or early diplotene) is highlighted by the dotted arc, the mid pachytene region is highlighted by * and late pachytene by **. All germlines are orientated the same way: distal to the left and proximal to the right.

Article Snippet: Western blot analysis was performed using ERK (K-23, Santa Cruz 1∶2000, rabbit) and phosphorylated ERK (clone MAPK-YT, Sigma, 1∶2000, mouse) specific antibodies that cross react with MPK-1 and phosphorylated MPK-1 , , or anti-GLD-1 (1∶500, rabbit, this study) and HRP conjugated secondary antibodies (anti-rabbit-HRP and anti-mouse-HRP, DakoCytomation 1∶2000).

Techniques: Irradiation, Immunofluorescence, Staining

Journal: Breast Cancer Research and Treatment

Article Title: Targeting ataxia telangiectasia-mutated- and Rad3-related kinase (ATR) in PTEN-deficient breast cancers for personalized therapy

doi: 10.1007/s10549-018-4683-4

Figure Lengend Snippet: Association between PTEN, ATR and pChk1expression in breast cancers

Article Snippet: A further set of slides were incubated for 60 min with the primary rabbit polyclonal anti-phosphorylated Chk1 antibody (Ab58567, Abcam, Cambridge, UK), at a dilution of 1:140 [ ].

Techniques:

Journal: Breast Cancer Research and Treatment

Article Title: Targeting ataxia telangiectasia-mutated- and Rad3-related kinase (ATR) in PTEN-deficient breast cancers for personalized therapy

doi: 10.1007/s10549-018-4683-4

Figure Lengend Snippet: a Nuclear and cytoplasmic staining of PTEN [a negative staining for both, b negative staining for nuclei and weak for cytoplasm, c some weak staining for nuclei and moderate for cytoplasm d strong staining for both; TMA cores pictures were taken using digital pathology interference at ×100 (left) and ×200 (right)]. Kaplan–Meier plot showing breast cancer-specific survival (BCSS) and b nuclear PTEN level. c combined nuclear PTEN and ATR level. d combined nuclear PTEN and cytoplasmic pCHK1 level. e combined ATR and cytoplasmic pCHK1 level nuclear PTEN negative tumours

Article Snippet: A further set of slides were incubated for 60 min with the primary rabbit polyclonal anti-phosphorylated Chk1 antibody (Ab58567, Abcam, Cambridge, UK), at a dilution of 1:140 [ ].

Techniques: Staining, Negative Staining

Journal: BMC Musculoskeletal Disorders

Article Title: Influence of Angptl1 on osteoclast formation and osteoblastic phenotype in mouse cells

doi: 10.1186/s12891-021-04278-6

Figure Lengend Snippet: Effects of Angptl1 on osteoclast formation in mouse bone marrow cells. a Mouse bone marrow cells were pre-cultured with 50 ng/ml of M-CSF for 3 days, and further cultured with 50 ng/ml of RANKL and M-CSF in the presence or absence of recombinant Angptl1 (100 ng/ml) for additional 4 days. The number of TRAP-positive MNCs was counted. Data represent mean ± SEM ( n = 6 in each group). ** p < 0.01 by Tukey-Kramer test. b Mouse bone marrow cells were pre-cultured with 50 ng/ml of M-CSF for 3 days, and further cultured with 50 ng/ml of RANKL and M-CSF in the presence or absence of recombinant Angptl1 (100 ng/ml) for additional 4 days. Then, total RNA was extracted for qPCR analysis of TRAP, cathepsin K (Ctsk), NFATc1, MMP-9, DC-STAMP or GAPDH. Data are expressed relative to GAPDH mRNA value. Data represent mean ± SEM (n = 6 in each group). Two independent experiments were performed. c Mouse bone marrow cells were pre-cultured with 50 ng/ml of M-CSF for 3 days, and then cultured with or without recombinant Angptl1 (100 ng/ml) for 1 h in the presence of 50 ng/ml of RANKL and M-CSF. Total proteins were extracted from the cells and Western blotting analyses for phosphorylated-p65 (p-p65), p65 and GAPDH were performed. The images represent experiments performed independently 3 times. Cont; Control

Article Snippet: Anti-phosphorylated p65 (Cat. No., 3033), anti-p65 (Cat. No., 8242) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cat. No. 5174) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Cell Culture, Recombinant, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual-Function Semaphorin 4D Released by Platelets: Suppression of Osteoblastogenesis and Promotion of Osteoclastogenesis

doi: 10.3390/ijms23062938

Figure Lengend Snippet: The effect of Sema4D and IGF-1 derived from TPs on ERK and Akt phosphorylation in MC3T3-E1 cells. Western blotting was performed to detect total and phosphorylated ERK and Akt in MC3T3-E1 cells cultured with/without ant-Sema4D mAb (50 µg/mL) or control mAb (50 µg/mL) in the presence of TPs ( A ) and total and phosphorylated ERK and Akt in MC3T3-E1 cells cultured with/without ant-IGF-1 mAb (1 µg/mL) in the presence of TPs ( B ). ALP activity was measured in TP-mediated MC3T3-E1 cells with and without ERK or Akt inhibitor ( C ). The mRNA expression of PlexinB1, PlexinB2 and CD72 in MC3T3-E1 cells was evaluated by qPCR ( D ). Data represent the mean ± SD of three independent experiments. Values are mean ± S.D. * p < 0.05, ** p < 0.01. +: Applied. −: Not applied.

Article Snippet: After transferal to a hydrophilic polyvinylidene fluoride (PVDF) membrane, the blot was probed overnight at 4 °C with mouse anti-Sema4D, rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-total ERK monoclonal antibody (Cell Signaling Technology), rabbit anti-phosphorylated ERK monoclonal antibody (Cell Signaling Technology), mouse anti-total Akt monoclonal antibody (Cell Signaling Technology) or mouse anti-phosphorylated Akt monoclonal antibody (Cell Signaling Technology).

Techniques: Derivative Assay, Phospho-proteomics, Western Blot, Cell Culture, Control, Activity Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: TSG-6 attenuates inflammation-induced brain injury via modulation of microglial polarization in SAH rats through the SOCS3/STAT3 pathway

doi: 10.1186/s12974-018-1279-1

Figure Lengend Snippet: The effects of rh-TSG-6 on phosphorylation of Stat3 and cellular location of p-STAT3. The phosphorylation of STAT3 was assessed by immunofluorescent double-labeling. Immunoreactivity for p-STAT3 is attenuated at 24 h in the SAH + rh-TSG-6 group, as compared with the SAH + vehicle group. TSG-6 deficiency increased the expression of p-STAT3 and more translocation from the cytosol to the nucleus occurs was observed. Scale bar = 20 μm

Article Snippet: The following primary antibodies were used for WB: mouse anti-TSG-6 (Santa Cruz Biotechnology; 1:800), rabbit anti-STAT3 (Cell Signaling Technology; 1:2000), rabbit anti-phosphorylated STAT3 at Tyr705 (Cell Signaling Technology; 1:1000), rabbit anti-SOCS3 (Abcam; 1:1000), mouse anti-CD163 (AbD Serotec; 1:500), rabbit anti-CD86 (ProteinTech; 1:600), rabbit anti-IL-6 (PeproTech; 1:800), rabbit anti-IL-10 (ProteinTech; 1:600), and rabbit anti-β-actin (Cell Signaling Technology; 1:1000).

Techniques: Phospho-proteomics, Labeling, Expressing, Translocation Assay

Journal: Journal of Neuroinflammation

Article Title: TSG-6 attenuates inflammation-induced brain injury via modulation of microglial polarization in SAH rats through the SOCS3/STAT3 pathway

doi: 10.1186/s12974-018-1279-1

Figure Lengend Snippet: Effects of rh-TSG-6 and TSG-6 siRNA treatments on SOCS3/STAT3 axis. a Effect of exogenous TSG-6 on STAT3 pathway expression was evaluated by western blot and the relative IL-6, IL-10, SOCS3, p-STAT3, and STAT3 densities were quantificationally evaluated. b The representative western blot analysis of STAT3 axis in SAH rats accepted intracerebroventricular injection of siRNA as indicated and quantification of the level of IL-6, IL-10, SOCS3, p-STAT3 and STAT3 after deficiency of TSG-6. Data are expressed as the mean ± SD from six rats. ** P < 0.01

Article Snippet: The following primary antibodies were used for WB: mouse anti-TSG-6 (Santa Cruz Biotechnology; 1:800), rabbit anti-STAT3 (Cell Signaling Technology; 1:2000), rabbit anti-phosphorylated STAT3 at Tyr705 (Cell Signaling Technology; 1:1000), rabbit anti-SOCS3 (Abcam; 1:1000), mouse anti-CD163 (AbD Serotec; 1:500), rabbit anti-CD86 (ProteinTech; 1:600), rabbit anti-IL-6 (PeproTech; 1:800), rabbit anti-IL-10 (ProteinTech; 1:600), and rabbit anti-β-actin (Cell Signaling Technology; 1:1000).

Techniques: Expressing, Western Blot, Injection