Journal: Clinical Interventions in Aging

Article Title: Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington’s disease via suppressing oxidative stress, inflammation, and apoptosis

doi: 10.2147/cia.s86493

Figure Lengend Snippet: Figure 7 Expressions of calpain and caspase 12, two markers of ER stress-related apoptosis, in the heart tissue of R6/2 HD mice under oral B307 treatment, and of their WT. Notes: (A) IHC staining illustrates that expressions of calpain and caspase 12 (shown in brown) were remarkable in comparison to their WT but were reduced under oral B307 treatment. Scale bars: 25 µm. (B) Western blotting analysis shows the following: (a) Cardiac expression levels of calpain and caspase 12 in R6/2 HD mice under sham and oral B307 treatments, and of their WT. (b) Quantified cardiac calpain and caspase 12 levels in R6/2 HD mice were significantly enhanced in comparison to their WT but were significantly weaker under oral B307 treatment. There were six mice per each group. Values are mean ± SEM (**P,0.01, *P,0.05, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest). Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; ER, endoplasmic reticulum; IHC, immunohistochemical; SEM, standard error of the mean; ANOVA, analysis of variance.

Article Snippet: This study used antibodies such as b-actin (Thermo Fisher Scientific Inc.), TNF-α (Cat #11948, Cell Signaling Technology Inc.), SOD2 (Cat #13141, Cell Signaling Technology Inc.), 3-NT (Cat ab61392, Abcam Inc.), 4-HNE (Cat ab46545, Abcam Inc.), Bcl-2 (Cat sc-7382, Santa Cruz Biotechnology Inc.), Bax (Cat #MS-714, Thermo Fisher Scientific Inc.), Cyto-C (Cat #1896-1, Abcam Inc.), calpain (Cat #2539, Cell Signaling Technology Inc.), caspase 12 (Cat #2202, Cell Signaling Technology Inc.), caspase 9 (Cat #9504, Cell Signaling Technology Inc.), and caspase 3 (Cat #9662, Cell Signaling Technology Inc.) to identify expression levels of these proteins in cardiac tissue by means of a horseradish peroxidase-linked secondary antibody.

Techniques: Immunohistochemistry, Comparison, Western Blot, Expressing, Immunohistochemical staining

Journal: Clinical Interventions in Aging

Article Title: Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington’s disease via suppressing oxidative stress, inflammation, and apoptosis

doi: 10.2147/cia.s86493

Figure Lengend Snippet: Figure 9 The schematic diagram illustrates the possible cardioprotective pathways under oral B307 treatment. Notes: Cardioprotection in R6/2 HD mice under B307 treatment may be promoted via attenuating oxidative stress (marked by increasing expression of SOD2), anti- apoptosis (marked by increasing expression of Bcl-2), reducing mHtt aggregation, oxidative stress (marked by reducing expressions of 3-NT and 4-HNE), inflammation (marked by reducing expression of TNF-α), ER stress-related apoptosis (marked by reducing expressions of calpain and caspase 12), and mitochondrial dysfunction-related apoptosis (marked by reducing expressions of Bax, Cyto-C, caspase 9, and caspase 3) in heart tissue. Abbreviations: mHtt, mutant huntingtin; SOD2, superoxide dismutase 2; ROS, reactive oxygen species; 3-NT, neurotrophin-3; 4-HNE, 4-hydroxynonenal; TNF-α, tumor necrosis factor alpha; ER, endoplasmic reticulum; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Cyto-C, cytochrome C; ER, endoplasmic reticulum; WT, wild-type littermate controls; HD, Huntington’s disease.

Article Snippet: This study used antibodies such as b-actin (Thermo Fisher Scientific Inc.), TNF-α (Cat #11948, Cell Signaling Technology Inc.), SOD2 (Cat #13141, Cell Signaling Technology Inc.), 3-NT (Cat ab61392, Abcam Inc.), 4-HNE (Cat ab46545, Abcam Inc.), Bcl-2 (Cat sc-7382, Santa Cruz Biotechnology Inc.), Bax (Cat #MS-714, Thermo Fisher Scientific Inc.), Cyto-C (Cat #1896-1, Abcam Inc.), calpain (Cat #2539, Cell Signaling Technology Inc.), caspase 12 (Cat #2202, Cell Signaling Technology Inc.), caspase 9 (Cat #9504, Cell Signaling Technology Inc.), and caspase 3 (Cat #9662, Cell Signaling Technology Inc.) to identify expression levels of these proteins in cardiac tissue by means of a horseradish peroxidase-linked secondary antibody.

Techniques: Expressing, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: The Phosphorylation State of GluR1 Subunits Determines the Susceptibility of AMPA Receptors to Calpain Cleavage

doi: 10.1074/jbc.m701283200

Figure Lengend Snippet: FIGURE 1. Activation of calpain causes the down-regulation of AMPAR currents in cortical pyramidal neurons. A, immunochemical images showing the co-staining of -calpain and MAP2 in cortical neurons transfected with calpain siRNA or a scrambled siRNA. B, plot of normalized peak AMPAR currents (IAMPA) with a prolonged NMDA application (100 M, 10 min) in GFP-positive neurons transfected with calpain siRNA or a scrambled siRNA. Inset, representative current traces taken at indicated time points. Scale bars, 100 pA, 1 s. C, cumulative data (mean S.E.) showing the percentage reduction of AMPAR currents by prolonged NMDA treatment in GFP-positive cells transfected with GFP alone, a scrambled siRNA or calpain siRNA. *, p 0.001, ANOVA.

Article Snippet: The calpain siRNA oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) contained three sense strands: 5 -CCACGUAGUCAUUACUCUA-3 , 5 - GACUAUCGGUAGCCAUGAA-3 , and 5 -GUACCCAGCUUCCCAAUCA-3 .

Techniques: Activation Assay, Staining, Transfection

Journal: Journal of Biological Chemistry

Article Title: The Phosphorylation State of GluR1 Subunits Determines the Susceptibility of AMPA Receptors to Calpain Cleavage

doi: 10.1074/jbc.m701283200

Figure Lengend Snippet: FIGURE 2. Calpain activation causes the cleavage of GluR1 subunits in cortical pyramidal neurons. A, Western blot analysis of various proteins in cultured cortical neurons transfected with calpain siRNA or a scrambled siRNA following glutamate (500 M, 10 min) or NMDA treatment (100 M, 10 min). Cells were collected after 10-min of washing. Antibodies against the N-terminal (NT) or C-terminal (CT) residues of GluR1, GluR2, or GluR2/3 were used. B, quantificative analysis (mean S.E.) showing the levels of GluR1 (detected with the N-terminal antibody) and GluR2 (detected with the N-ter- minal antibody) with glutamate or NMDA treatment in cortical cultures trans- fected with calpain siRNA or a scrambled siRNA. *, p 0.001, ANOVA.

Article Snippet: The calpain siRNA oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) contained three sense strands: 5 -CCACGUAGUCAUUACUCUA-3 , 5 - GACUAUCGGUAGCCAUGAA-3 , and 5 -GUACCCAGCUUCCCAAUCA-3 .

Techniques: Activation Assay, Western Blot, Cell Culture, Transfection

Journal: Journal of Biological Chemistry

Article Title: The Phosphorylation State of GluR1 Subunits Determines the Susceptibility of AMPA Receptors to Calpain Cleavage

doi: 10.1074/jbc.m701283200

Figure Lengend Snippet: FIGURE 3. Inhibition of CaMKII attentuates the effect of prolonged NMDA treatment on AMPAR currents. A, plot of normalized IAMPA with a prolonged NMDA application (100 M, 10 min) in the absence (control) or presence of KN-93 (20 M). B, cumulative data (mean S.E.) showing the percentage reduction of AMPAR currents by prolonged NMDA treatment with different agents that affect CaMKII activity. AIP, autocamtide-2-related inhibitory pep- tide. C, plot of normalized IAMPA in GFP-positive neurons transfected with or without CaMKII siRNA. D, cumulative data (mean S.E.) summarizing the percentage reduction of AMPAR currents by prolonged NMDA treatment in GFP-positive cells transfected with CaMKII siRNA or a scrambled siRNA. E, plot of IAMPA showing the effect of KN-93 (20 M, 10 min) on AMPAR currents. F, plot of IAMPA showing the effect of a prolonged NMDA application (100 M, 10 min) and a subsequent application of KN-93 (20 M). Inset (A, C, E, and F), representative current traces taken from the recordings at indicated times. Scale bars, 100 pA, 1 s. *, p 0.001, ANOVA.

Article Snippet: The calpain siRNA oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) contained three sense strands: 5 -CCACGUAGUCAUUACUCUA-3 , 5 - GACUAUCGGUAGCCAUGAA-3 , and 5 -GUACCCAGCUUCCCAAUCA-3 .

Techniques: Inhibition, Control, Activity Assay, Transfection

Journal: Journal of Biological Chemistry

Article Title: The Phosphorylation State of GluR1 Subunits Determines the Susceptibility of AMPA Receptors to Calpain Cleavage

doi: 10.1074/jbc.m701283200

Figure Lengend Snippet: FIGURE 4. Phosphorylation of GluR1 by CaMKII at Ser831 determines the susceptibility of GluR1 to calpain cleavage. A, immunoblots of the GluR1 fusion protein treated with calpain (5 units/ml, 30 min) in the absence or presence of different agents that affect calpain cleavage (added 10 min before calpain treatment), including 11R-CS (10 M), CaMKII (0.6 g/ml), and KN-93 (20 M). GluR1 was detected with an antibody against hemagglutinin, which labeled both the cleaved and uncleaved GluR1 fusion protein. B, quantitative analysis (means S.E.) showing the normalized level of calpain-cleaved GluR1 fusion protein in the presence of various agents. *, p 0.001, ANOVA. C, immunoblots of different GluR1 fusion proteins (con- trol,S831A,S831D,S831E,S845A)treatedwithcalpain(5units/ml,30min)intheabsenceorpresenceofCaMKII (0.6 g/ml, added 10 min before calpain treatment). D, quantitative analysis (means S.E.) showing the normalized level of calpain-cleaved different GluR1 fusion proteins in the absence or presence of CaMKII. *, p 0.001, ANOVA, compared with control (CaMKII).

Article Snippet: The calpain siRNA oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) contained three sense strands: 5 -CCACGUAGUCAUUACUCUA-3 , 5 - GACUAUCGGUAGCCAUGAA-3 , and 5 -GUACCCAGCUUCCCAAUCA-3 .

Techniques: Phospho-proteomics, Western Blot, Labeling, Control

Journal: Molecular oncology

Article Title: Growth arrest-specific protein 2 (GAS2) interacts with CXCR4 to promote T-cell leukemogenesis partially via c-MYC.

doi: 10.1002/1878-0261.13306

Figure Lengend Snippet: Fig. 3. GAS2 interacts with CXCR4. (A) The expression of CXCR4 in Calpain2 silenced (shCPN2) and control (Scrambled) Jurkat cells was analyzed by western blot (n = 3). (B) The protein extraction of Jurkat cells was prepared. An aliquot of the extract was used as an untreated control. One aliquot of the extract was supplied with Ca++ only, and the other aliquot was added with both Ca++ and Calpain inhibitor (Z-LLY- FMK); both aliquots were incubated at 37 °C for 8 h. Finally, the three aliquots of lysates were separated with SDS/PAGE and transferred onto a PVDG membrane. Ponceau S staining was used to monitor the loading of the samples. The expression of CXCR4 and GAPDH was analyzed by western blot. (n = 4) (C) The dominant negative form of GAS2 (GAS2DN) was overexpressed in Jurkat cells, the expression of CXCR4 in these cells together with their control was analyzed by western blot (n = 3). (D) Calpain2 was silenced in GAS2DN overexpressed Jurkat cells, and then CXCR4 expression in these cells together with the control cells was analyzed by western blot (n = 3). (E) Flag-tagged GAS2 and/or HA tagged CXCR4 were overexpressed in 293 T cells, and coimmunoprecipitation (Co-IP) experiment was performed to ana- lyze the interaction between GAS2 and CXCR4 (n = 2). (F) Immunoprecipitation (IP) against GAS2 in Jurkat cells was performed (left panel), and the expression of CXCR4 and Calpain2 (CPN2) was detected with immunoblot (IB). Conversely, IP against CXCR4 in Jurkat cells was conducted (right panel), and the expression of GAS2 and CPN2 was detected with IB (n = 2). (G) The expression of both GAS2 (green) and CXCR4 (red) was analyzed with confocal microscopy (n = 2). (H) Additionally, the expression of both Calpain2 (green) and CXCR4 (red) was analyzed with confocal microscopy (n = 2). The scale bar equals 5 or 20 lm as indicated in each graph.

Article Snippet: Briefly, the cells were first incubated with antibody against GAS2 (ab109762; Abcam, Waltham, MA, USA) or Calpain2 (A4066; ABclonal, Wuhan, China), and they were incubated with antibody against CXCR4 (60042-1-Ig; Proteintech).

Techniques: Expressing, Control, Western Blot, Protein Extraction, Incubation, SDS Page, Membrane, Staining, Dominant Negative Mutation, Co-Immunoprecipitation Assay, Immunoprecipitation, Confocal Microscopy