Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Modulation of the voltage-gated potassium channel Kv1.5 by the SGK1 protein kinase involves inhibition of channel ubiquitination.

doi: 10.1159/000185543

Figure Lengend Snippet: Fig. 4. Expression of the ubiquitin ligase Nedd4-2 reduces Kv1.5 activity by ubiquitinating and thereby inhibiting Kv1.5 plasma membrane expression. Xenopus oocytes were injected with wild-type Kv1.5 alone or together with the ubiquitin ligase Nedd4-2. Four days after cRNA injection, voltage-activated currents were measured and normalized in each batch of oocytes to the mean value obtained in oocytes expressing wild-type Kv1.5 alone (Arithmetic means ± SEM, * indicates statistically significant difference to currents in Xenopus oocytes expressing Kv1.5 alone) (A). In addition, Kv1.5 ubiquitination was assessed in whole cell lysates (B) and in isolated plasma membrane fraction after channel immunoprecipitation (C, upper panel) or by using the UbiQaptureTM-Q kit (C, lower panel). Kv1.5 protein abundance was determined by western blotting of isolated plasma membranes (D).

Article Snippet: After blocking with 5 % non-fat dry milk in PBS/ 0.15 % Tween 20 for 1h at room temperature, blots were incubated overnight at 4 °C with a rabbit anti-SGK1 antibody (Upstate, Waltham, MA, USA, diluted 1:2000 in PBS/0.15 % Tween 20/5 % non-fat dry milk), a rabbit anti-phospho (Ser/ Thr) antibody 110B7E (Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000 in TBS/0.15 % Tween 20/5 % BSA) or a mouse anti-ubiquitin antibody (Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000 in TBS/0.15 % Tween 20/5 % non-fat dry milk).

Techniques: Expressing, Ubiquitin Proteomics, Activity Assay, Clinical Proteomics, Membrane, Injection, Isolation, Immunoprecipitation, Quantitative Proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Experimental protocol and changes in HMGB1 levels in the brain and plasma 48 h after the EDH. ( A ) Experimental protocol of the EDH. ( B ) Typical appearance picture of the EDH. The brain samples were collected 48 h after the induction of the EDH from the dotted area in ( B ) and were used for western blotting for HMGB1. ( C ) The representative results of western blotting are shown. A decrease in HMGB1 levels in the ipsilateral cortex (peri-hematoma) in the control IgG group is shown. ( D ) Quantitative analyses of western blotting results were performed using NIH Image J software V.1.53, and the results are expressed as mean ± SE of 3–5 rats per group. Differences were considered significant at # p < 0.05 compared to the sham group. * p < 0.05 compared to the control IgG-treated group. ( E ) Plasma levels of HMGB1 in rats with an EDH were determined by ELISA. Blood samples were collected 48 h after the induction of hemorrhage. The results are expressed as mean ± SE of 3–5 rats per group. # p < 0.05 compared to the sham group. * p < 0.05 compared to the control IgG-treated group.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Western Blot, Software, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Effects of the anti-HMGB1 mAb on the expression of inflammation-related molecules and apoptosis 48 h after the EDH. ( A ) The mRNA expression of TNF-α, iNOS, and IL-1β was measured by quantitative real-time polymerase chain reaction in the cerebral cortex (peri-hematoma) 48 h after the EDH. The primer information refers to previous research . The results are expressed as mean ± SE of 5 rats ( A ). # p < 0.05 compared to the sham group. * p < 0.05 and compared to the control IgG-treated group. ( B ) TUNEL staining was performed to reveal apoptotic cells in the ipsilateral cerebral cortex in each group. Scale bar: 50 μm.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Expression of AQP4, PAR1, PAI-1, and MSR1 after the EDH and effects of anti-HMGB1 treatment. ( A ) The protein levels of AQP4 and PAR-1 in the ipsilateral cerebral cortex (beneath the hematoma) were determined by western blotting 48 h after the EDH. The representative results of western blotting are shown. ( B ) The quantitative results are expressed as mean ± SE of 3–5 rats. The protein levels of AQP4 and PAR-1 in the ipsilateral cerebral cortex (beneath the hematoma) were determined by western blotting 48 h after the EDH. ## p < 0.01, and ### p < 0.001 compared to the sham group. * p < 0.05 compared to the control IgG-treated group. ns means no significant difference. ( C ) The protein levels of PAI-1 and MSR-1 in the ipsilateral cerebral cortex (beneath the hematoma) were determined by western blotting 48 h after the EDH. The representative results of western blotting are shown. ( D ) The quantitative results are expressed as mean ± SE of 3–5 rats. The protein levels of PAI-1 and MSR1 in the ipsilateral cerebral cortex (beneath the hematoma) were determined by western blotting 48 h after the EDH. # p < 0.05, and ### p < 0.001 compared to the sham group. ** p < 0.01, and *** p < 0.01 compared to the control IgG-treated group.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Effects of the anti-HMGB1 mAb on stimulant-induced HMGB1 translocation in neuroblastoma cells in culture. ( A ) SH-SY5Y neuroblastoma cells were stimulated with hemin (1 mM) for 24 h, and the translocation of HMGB1 was observed immunocytochemically. The anti-HMGB1 mAb (10 μg/mL) or anti-KLH mAb (control) was added to the media immediately before the start of incubation. DAPI was used for nuclear staining. Scale bar: 20 μm. ( B ) IL-6 and IL-8 levels in the media stimulated with hemin (0.6 mM) were determined 24 h after stimulation in the presence (10 μg/mL) or absence of the anti-HMGB1 mAb. The results are expressed as mean ± SE of triplicate experiments. * p < 0.05 and ** p < 0.01 compared to the non-stimulated group or to the control IgG-treated group, as shown by the lines in the figure. ( C ) SH-SY5Y neuroblastoma cells were stimulated with FeCl 2 (0.6 mM) for 24 h in the presence or absence of the anti-HMGB1 mAb (10 μg/mL), and the translocation of HMGB1 was observed immunocytochemically. Scale bar: 20 μm. ( D ) SH-SY5Y neuroblastoma cells were stimulated with thrombin (5 U/mL) for 24 h in the presence or absence of the anti-HMGB1 mAb (10 μg/mL), and the translocation of HMGB1 was observed immunocytochemically. Scale bar: 20 μm.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Translocation Assay, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Anti-HMGB1 mAb Therapy Reduces Epidural Hematoma Injury

doi: 10.3390/ijms25115889

Figure Lengend Snippet: Effects of the anti-HMGB1 mAb on TNF-α-induced HMGB1 translocation in vascular endothelial cells in culture. EA.hy 926 vascular endothelial cells were stimulated with TNF (100 ng/mL) in the presence or absence of the anti-HMGB1 mAb (10 μg/mL) for 6 h, and the translocation of HMGB1 was observed immunocytochemically. DAPI was used for nuclear staining.

Article Snippet: The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4 °C with primary antibodies against HMGB1 (ab18256, Abcam, Waltham, MA, USA), Heat Shock Protein 27 (Hsp27) (ab2790, Abcam, Waltham, MA, USA), Hsp40 (ab69402, Abcam, Waltham, MA, USA), Hsp60 (ab46798, Abcam, Waltham, MA, USA), Hsp90 (ab13492, Abcam, Waltham, MA, USA), Peroxiredoxin 5 (Prx5) (ab180587, Abcam, Waltham, MA, USA), Prx6 (ab195045, Abcam, Waltham, MA, USA), Aquaporin-4 (AQP4) (ab9512, Abcam, Waltham, MA, USA), Protease-Activated Receptor 1 (PAR1) (PA5-116040, Invitrogen, Carlsbad, CA, USA), and Plasminogen Activator Inhibitor-1 (PAI-1) (ab66705, Abcam, Waltham, MA, USA). β-actin (sc-47778, Santa Cruz, CA, USA) was used as a reference protein.

Techniques: Translocation Assay, Staining

Journal: Bioengineered

Article Title: Binding and biologic characterization of recombinant human serum albumin-eTGFBR2 fusion protein expressed in CHO cells

doi: 10.1080/21655979.2017.1292186

Figure Lengend Snippet: The Smad3 activation analysis of HSC-T6 cells. Cells were incubated with 30 nM eTGFBR2 (A) or HSA-eTGFBR2 (B) for 0, 0.5, 1, 2 h, and then exposed to 0.4 nM TGF-β1 for 30 min. -: DMEM medium for negative control; +: 0.4 nM TGF-β1 for positive control.

Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with anti-p-Smad3 antibody or anti-Smad3 antibody (Cell Signaling technology) for 4 h at room temperature.

Techniques: Activation Assay, Incubation, Negative Control, Positive Control