calpain reaction buffer  (Millipore)


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    Structured Review

    Millipore calpain reaction buffer
    Schematic diagram showing the relationship of <t>calpain/NF-κB/inflammation/NVU</t> damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
    Calpain Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/calpain reaction buffer/product/Millipore
    Average 91 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    calpain reaction buffer - by Bioz Stars, 2020-05
    91/100 stars

    Images

    1) Product Images from "Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice"

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    Journal: Chinese Medical Journal

    doi: 10.4103/0366-6999.198001

    Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
    Figure Legend Snippet: Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Techniques Used: Mouse Assay, Activation Assay

    MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P
    Figure Legend Snippet: MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Techniques Used: Activity Assay, Expressing, Western Blot

    2) Product Images from "Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice"

    Article Title: Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddu112

    αSyn cleavage by calpain 1 in vitro . ( A ) Scheme of the human αSyn sequence indicating the cleavage sites of calpain 1 and the epitopes of the antibodies used in this study. The main cleavage sites (arrows) of calpain 1 are located in the
    Figure Legend Snippet: αSyn cleavage by calpain 1 in vitro . ( A ) Scheme of the human αSyn sequence indicating the cleavage sites of calpain 1 and the epitopes of the antibodies used in this study. The main cleavage sites (arrows) of calpain 1 are located in the

    Techniques Used: In Vitro, Sequencing

    Expression of calpain 1 is changed and decreases calpain-mediated spectrin breakdown product in a mouse model of Parkinson's disease upon calpastatin overexpression. ( A ) Lysates of the brain stem ( n = 3) were used for western blot analysis. Decreased
    Figure Legend Snippet: Expression of calpain 1 is changed and decreases calpain-mediated spectrin breakdown product in a mouse model of Parkinson's disease upon calpastatin overexpression. ( A ) Lysates of the brain stem ( n = 3) were used for western blot analysis. Decreased

    Techniques Used: Expressing, Over Expression, Western Blot

    3) Product Images from "Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury"

    Article Title: Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2826-07.2007

    Calpastatin overexpression inhibits AIF translocation in CA1 neurons after global ischemia. A , Overexpression of calpastatin (Cps) after AAV infection in the rat brain. AAV was infused into the hippocampus using convection methodology to achieve widespread expression in the CA1 sector. Images show HA immunoreactivity (HA–Cps) at 14 d after AAV–Cps infection ( b ); preabsorption of the antibody with Cps protein abolished the signals ( c ). No Cps immunoreactivity is seen in CA1 after empty vector infection ( a ). Western blots (anti-HA or anti-Cps) confirm the expression of Cps protein in the CA1 sector at 7–14 d after AAV–Cps infection; n = 5 per group. B , C , AAV–Cps infection inhibited global ischemia-induced calpain activation in CA1. Western blot ( B ) was performed at 36 h after ischemia to detect α-fodrin cleavage, which was inhibited by AAV–Cps (lanes 6, 7) but not by empty AAV (lanes 4, 5). Substrate-based activity assays ( C ) show that ischemia-induced increases in calpain activity were inhibited by AAV–Cps. * p
    Figure Legend Snippet: Calpastatin overexpression inhibits AIF translocation in CA1 neurons after global ischemia. A , Overexpression of calpastatin (Cps) after AAV infection in the rat brain. AAV was infused into the hippocampus using convection methodology to achieve widespread expression in the CA1 sector. Images show HA immunoreactivity (HA–Cps) at 14 d after AAV–Cps infection ( b ); preabsorption of the antibody with Cps protein abolished the signals ( c ). No Cps immunoreactivity is seen in CA1 after empty vector infection ( a ). Western blots (anti-HA or anti-Cps) confirm the expression of Cps protein in the CA1 sector at 7–14 d after AAV–Cps infection; n = 5 per group. B , C , AAV–Cps infection inhibited global ischemia-induced calpain activation in CA1. Western blot ( B ) was performed at 36 h after ischemia to detect α-fodrin cleavage, which was inhibited by AAV–Cps (lanes 6, 7) but not by empty AAV (lanes 4, 5). Substrate-based activity assays ( C ) show that ischemia-induced increases in calpain activity were inhibited by AAV–Cps. * p

    Techniques Used: Over Expression, Translocation Assay, Infection, Convection, Expressing, Plasmid Preparation, Western Blot, Activation Assay, Activity Assay

    Calpain inhibition prevents OGD-induced AIF release in neurons. A , Primary neurons were infected for 3 d with empty AAV or AAV carrying the calpastatin cDNA (AAV–Cps), and the expression of Cps (tagged with triple HA) was confirmed by Western blot using antibodies against Cps and HA, respectively. B , Neurons infected with empty AAV or AAV–Cps were subjected to OGD (60 min), and Western blot was performed to detect α-fodrin cleavage, a marker for calpain activation, at 2 and 6 h after OGD. C , Neurons infected with empty AAV or AAV–Cps were subjected to OGD (60 min), and calpain activity was measured using substrate-based assays at 2, 6, and 16 h after OGD, and the data are expressed as fold increase over non-OGD controls (Con). * p
    Figure Legend Snippet: Calpain inhibition prevents OGD-induced AIF release in neurons. A , Primary neurons were infected for 3 d with empty AAV or AAV carrying the calpastatin cDNA (AAV–Cps), and the expression of Cps (tagged with triple HA) was confirmed by Western blot using antibodies against Cps and HA, respectively. B , Neurons infected with empty AAV or AAV–Cps were subjected to OGD (60 min), and Western blot was performed to detect α-fodrin cleavage, a marker for calpain activation, at 2 and 6 h after OGD. C , Neurons infected with empty AAV or AAV–Cps were subjected to OGD (60 min), and calpain activity was measured using substrate-based assays at 2, 6, and 16 h after OGD, and the data are expressed as fold increase over non-OGD controls (Con). * p

    Techniques Used: Inhibition, Infection, Expressing, Western Blot, Marker, Activation Assay, Activity Assay

    Truncation of AIF enables its disassociation with HSP10 in the mitochondria. A , HSP10 is an endogenous binding protein for AIF in neurons. Left, Proteins from cultured neurons were first immunoprecipitated with anti-HSP60 or anti-HSP10 antibody and then immunoblotted with anti-AIF, anti-HSP60, and anti-HSP10 antibodies. Right, Proteins were first immunoprecipitated with anti-AIF or anti-HSP60 antibody and then immunoblotted with anti-AIF, anti-HSP60, and anti-HSP10 antibodies. B , Autoradiograph shows direct protein–protein interaction between AIF and HSP10. The protein-binding assay was performed using in vitro translated proteins (the Flag–HSP10 and [ 35 S]AIF). The reaction products were immunoprecipitated using the anti-Flag antibody, electrophoresed, and then autoradiographed. C , Autoradiograph for [ 35 S]AIF shows that Flag–HSP10 can directly bind to AIF 62 but not to AIF 57 and that calpain I, which cleaves AIF, inhibited AIF 62 /HSP10 binding but not the binding between HSP10 and the calpain-resistant AIFm. The protein-binding assay was performed as described in B . D , Western blots show decreased AIF/HSP10 binding after OGD in empty AAV-infected neurons. Cell extracts were immunoprecipitated using the anti-HSP10 antibody and then immunoblotted with the anti-AIF antibody. The AIF/HSP10 binding was restored in neurons transfected with AAV–Cps. The graph at the bottom illustrates the relative changes of three independent experiments. * p
    Figure Legend Snippet: Truncation of AIF enables its disassociation with HSP10 in the mitochondria. A , HSP10 is an endogenous binding protein for AIF in neurons. Left, Proteins from cultured neurons were first immunoprecipitated with anti-HSP60 or anti-HSP10 antibody and then immunoblotted with anti-AIF, anti-HSP60, and anti-HSP10 antibodies. Right, Proteins were first immunoprecipitated with anti-AIF or anti-HSP60 antibody and then immunoblotted with anti-AIF, anti-HSP60, and anti-HSP10 antibodies. B , Autoradiograph shows direct protein–protein interaction between AIF and HSP10. The protein-binding assay was performed using in vitro translated proteins (the Flag–HSP10 and [ 35 S]AIF). The reaction products were immunoprecipitated using the anti-Flag antibody, electrophoresed, and then autoradiographed. C , Autoradiograph for [ 35 S]AIF shows that Flag–HSP10 can directly bind to AIF 62 but not to AIF 57 and that calpain I, which cleaves AIF, inhibited AIF 62 /HSP10 binding but not the binding between HSP10 and the calpain-resistant AIFm. The protein-binding assay was performed as described in B . D , Western blots show decreased AIF/HSP10 binding after OGD in empty AAV-infected neurons. Cell extracts were immunoprecipitated using the anti-HSP10 antibody and then immunoblotted with the anti-AIF antibody. The AIF/HSP10 binding was restored in neurons transfected with AAV–Cps. The graph at the bottom illustrates the relative changes of three independent experiments. * p

    Techniques Used: Binding Assay, Cell Culture, Immunoprecipitation, Autoradiography, Protein Binding, In Vitro, Western Blot, Infection, Transfection

    The wild-type AIF, but not the calpain-resistant mutant AIF, restores OGD-induced cell death of AIF-deficient neurons. A , B , Neurons from Hq mice or wild-type (Wt) mice were infected with AAV–AIF, AAV–AIFm, or empty AAV for 3 d and then subjected to OGD for 60 min. Cell viability ( A ) and cell death ( B ) were measured at 24 and 48 h after OGD using Alamar blue fluorescence and phosphatidylinositol/Hoechst 33258 staining, respectively. * p
    Figure Legend Snippet: The wild-type AIF, but not the calpain-resistant mutant AIF, restores OGD-induced cell death of AIF-deficient neurons. A , B , Neurons from Hq mice or wild-type (Wt) mice were infected with AAV–AIF, AAV–AIFm, or empty AAV for 3 d and then subjected to OGD for 60 min. Cell viability ( A ) and cell death ( B ) were measured at 24 and 48 h after OGD using Alamar blue fluorescence and phosphatidylinositol/Hoechst 33258 staining, respectively. * p

    Techniques Used: Mutagenesis, Mouse Assay, Infection, Fluorescence, Staining

    Calpain-dependent truncation of AIF is required for its release after OGD. A , Neurons were infected for 3 d with AAV carrying either the AIF-GFP fusion construct ( a–l ) or the calpain-resistant mutant (L101/103G) AIFm-GFP construct ( m–t ) and then subjected to OGD for 60 min. Confocal images were taken on triple-label immunofluorescent neurons (GFP, green; COX IV, red; Hoechst 33258, blue) in non-OGD cultures ( a–d , m–p ) or at 2 h ( e–h , q–t ) and 6 h ( i–l ) after OGD. In non-OGD neurons, AIF–GFP and AIFm–GFP are localized in mitochondria; after OGD, AIF–GFP is translocated into the nucleus ( e–l ), whereas AIFm–GFP remains in the mitochondria ( q–t ). B , Percentages of transfected neurons showing nuclear localization of AIF–GFP or AIFm–GFP at 2, 6, and 16 h after 60 min OGD. * p
    Figure Legend Snippet: Calpain-dependent truncation of AIF is required for its release after OGD. A , Neurons were infected for 3 d with AAV carrying either the AIF-GFP fusion construct ( a–l ) or the calpain-resistant mutant (L101/103G) AIFm-GFP construct ( m–t ) and then subjected to OGD for 60 min. Confocal images were taken on triple-label immunofluorescent neurons (GFP, green; COX IV, red; Hoechst 33258, blue) in non-OGD cultures ( a–d , m–p ) or at 2 h ( e–h , q–t ) and 6 h ( i–l ) after OGD. In non-OGD neurons, AIF–GFP and AIFm–GFP are localized in mitochondria; after OGD, AIF–GFP is translocated into the nucleus ( e–l ), whereas AIFm–GFP remains in the mitochondria ( q–t ). B , Percentages of transfected neurons showing nuclear localization of AIF–GFP or AIFm–GFP at 2, 6, and 16 h after 60 min OGD. * p

    Techniques Used: Infection, Construct, Mutagenesis, Transfection

    4) Product Images from "Altered Regulation of Striatal Neuronal N-Methyl-D-Aspartate Receptor Trafficking by Palmitoylation in Huntington Disease Mouse Model"

    Article Title: Altered Regulation of Striatal Neuronal N-Methyl-D-Aspartate Receptor Trafficking by Palmitoylation in Huntington Disease Mouse Model

    Journal: Frontiers in Synaptic Neuroscience

    doi: 10.3389/fnsyn.2019.00003

    Calpain cleavage of GluN2B to isolate Cys cluster II-containing C-terminal fragment reveals reduced palmitoylation in YAC128 striatum. (A) Cartoon indicating relative location of GluN2B Cys clusters I and II and the major calpain cleavage site. Each panel of image data shows results from one representative gel and the dividing line(s) indicate where lanes were removed for ease of comparing genotypes under identical experimental conditions. (B–D) Palmitoylation of Cys cluster II-containing C-terminal fragment was examined by IP/Btn-BMCC method. Representative blots show that palmitoylation (detected by probing with streptavidin Alexa 680, B ) of GluN2B Cys cluster II-containing C-terminal fragment as a ratio to total GluN2B levels (detected by C-terminal specific antibody MA1-2014, as shown in C ) was significantly decreased in striatum from YAC128 compared to FVB/N wild-type mice. Black arrow = ∼62 kDa cluster II-containing C-terminal fragment. (D) Summary graph indicates the corresponding ratio of palmitoylation signal (calculated as in Figure 1 ) for YAC128 normalized to the FVB/N control. Data presented for striatal tissue from 10 independent experiments; bars represent means ± SD ∗ p
    Figure Legend Snippet: Calpain cleavage of GluN2B to isolate Cys cluster II-containing C-terminal fragment reveals reduced palmitoylation in YAC128 striatum. (A) Cartoon indicating relative location of GluN2B Cys clusters I and II and the major calpain cleavage site. Each panel of image data shows results from one representative gel and the dividing line(s) indicate where lanes were removed for ease of comparing genotypes under identical experimental conditions. (B–D) Palmitoylation of Cys cluster II-containing C-terminal fragment was examined by IP/Btn-BMCC method. Representative blots show that palmitoylation (detected by probing with streptavidin Alexa 680, B ) of GluN2B Cys cluster II-containing C-terminal fragment as a ratio to total GluN2B levels (detected by C-terminal specific antibody MA1-2014, as shown in C ) was significantly decreased in striatum from YAC128 compared to FVB/N wild-type mice. Black arrow = ∼62 kDa cluster II-containing C-terminal fragment. (D) Summary graph indicates the corresponding ratio of palmitoylation signal (calculated as in Figure 1 ) for YAC128 normalized to the FVB/N control. Data presented for striatal tissue from 10 independent experiments; bars represent means ± SD ∗ p

    Techniques Used: Mouse Assay

    5) Product Images from "Calcium Dysregulation Induces Apoptosis-inducing Factor Release: Cross-talk Between PARP-1- and Calpain- Signaling Pathways"

    Article Title: Calcium Dysregulation Induces Apoptosis-inducing Factor Release: Cross-talk Between PARP-1- and Calpain- Signaling Pathways

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2009.04.032

    Calpain inhibition prevents NMDA-induced AIF truncation and nuclear translocation in neurons
    Figure Legend Snippet: Calpain inhibition prevents NMDA-induced AIF truncation and nuclear translocation in neurons

    Techniques Used: Inhibition, Translocation Assay

    Attenuation of NMDA-induced AIF nuclear translocation in neurons by inhibiting calpain or PARP-1
    Figure Legend Snippet: Attenuation of NMDA-induced AIF nuclear translocation in neurons by inhibiting calpain or PARP-1

    Techniques Used: Translocation Assay

    Model of PARP-1 and calpain-mediated AIF release
    Figure Legend Snippet: Model of PARP-1 and calpain-mediated AIF release

    Techniques Used:

    Related Articles

    Incubation:

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice
    Article Snippet: .. [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6). .. The reaction was initiated by the addition of CaCl2 (final concentration of 5 μmol/L) and the mixture was incubated at 37°C for 30 min.

    Article Title: Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice
    Article Snippet: .. Briefly, 1.25 µg of recombinant soluble as well as fibrillar [WT]αSyn and [A30P]αSyn were incubated in a calpain reaction buffer containing 1000 ng calpain 1 at 37°C for 30 min. To verify the specific cleavage of αSyn by calpain, we utilized an inhibitor specific for calpain (ALLN, Sigma). .. The reaction was stopped by the addition of 5× Laemmli buffer and denaturation of samples at 95°C before western blot analysis.

    Article Title: Ethyl pyruvate protects against hypoxic-ischemic brain injury via anti-cell death and anti-inflammatory mechanisms
    Article Snippet: .. In brief, mitochondrial proteins (30 µg) were incubated with calpain reaction buffer (20 mM HEPES, pH 7.6; 1 mM EDTA; 50 mM NaCl; and 0.1% (v/v) 2-mercaptoethanol) containing 10 µM calpain I fluorescent substrate H-E(EDANS)PLFAERK(DABCYL)-OH (Calbiochem, La Jolla, CA). .. The reaction was initiated by addition of CaCl2 (final concentration of 5 µM) and incubated at 37 °C for 30 min.

    Article Title: Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury
    Article Snippet: .. In brief, whole-cell or mitochondrial proteins at the indicated amounts were incubated with calpain reaction buffer [20 m m HEPES, pH 7.6, 1 m m EDTA, 50 m m NaCl, and 0.1% (v/v) 2-mercaptoethanol] containing 10 μ m calpain I fluorescent substrate H-E(EDANS)PLF∼AERK(DABCYL)-OH (Calbiochem, La Jolla, CA). .. The reaction was initiated by addition of CaCl2 (final concentration of 5 μ m ) and incubated at 37°C for 30 min.

    Article Title: Calcium Dysregulation Induces Apoptosis-inducing Factor Release: Cross-talk Between PARP-1- and Calpain- Signaling Pathways
    Article Snippet: .. In brief, mitochondrial proteins (30 μg) were incubated with calpain reaction buffer [20 mM HEPES, pH 7.6, 1 mM EDTA, 50 mM NaCl, and 0.1% (v/v) 2-mercaptoethanol] containing 10 μM calpain I fluorescent substrate HE(EDANS)PLF AERK(DABCYL)-OH (Calbiochem, La Jolla, CA). .. The reaction was initiated by addition of CaCl2 (final concentration of 5 μM) and incubated at 37°C for 30 min.

    Recombinant:

    Article Title: Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice
    Article Snippet: .. Briefly, 1.25 µg of recombinant soluble as well as fibrillar [WT]αSyn and [A30P]αSyn were incubated in a calpain reaction buffer containing 1000 ng calpain 1 at 37°C for 30 min. To verify the specific cleavage of αSyn by calpain, we utilized an inhibitor specific for calpain (ALLN, Sigma). .. The reaction was stopped by the addition of 5× Laemmli buffer and denaturation of samples at 95°C before western blot analysis.

    Article Title: Altered Regulation of Striatal Neuronal N-Methyl-D-Aspartate Receptor Trafficking by Palmitoylation in Huntington Disease Mouse Model
    Article Snippet: .. In brief, immunoprecipitates from both portions were diluted in 400 μl calpain reaction buffer (20 mM HEPES pH7.6, 10 mM KCl, 1.5 mM MgCl2 , 1 mM dithiothreitol) with 10 nM of recombinant calpain-1 (EMD-Millipore, 208712) and 2 mM CaCl2 for 10 min at RT, except one half was pre-treated with 100 μM of calpain inhibitor CI-III (EMD Millipore, 208722). .. The calpain cleavage reactions were stopped by adding 100 μM of calpain inhibitor CI-III (EMD Millipore, 208722) and centrifuging immediately for 5 s at 13,200 rpm.

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    Millipore calpain reaction buffer
    Schematic diagram showing the relationship of <t>calpain/NF-κB/inflammation/NVU</t> damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
    Calpain Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/calpain reaction buffer/product/Millipore
    Average 91 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    calpain reaction buffer - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    90
    Millipore calpain cleavage buffer
    <t>Calpain</t> 2 is activated and aggravates I/R injury in fatty livers. ( a ) Lean and ob/ob mice were subjected to 1 h of ischemia and 6 h of reperfusion. Liver calpain 1 and calpain 2 expression levels were analyzed by western blotting. Quantification of the relative protein level is shown (right; n =4 per group). ( b ) Isolated hepatocytes from lean and ob/ob mice underwent 4 h of anoxia and 20 min of reoxygenation. Calpain 1 and calpain 2 expression levels were determined at each time point. Quantification of relative protein level is shown (right; n =4 per group). ( c ) Calpain activity was determined by SLLVY-AMC fluorometry at each time point. ( d ). Ob/ob mice were pretreated with calpain inhibitor III (10 mg/kg) or vehicle DMSO for 6 h and then subjected to 1-h ischemia and 24 h of reperfusion. Necrotic area of liver samples was identified under light microscope (200x). Quantification of the necrotic areas is shown (right; n =4). ( e and f ) Isolated hepatocytes from ob/ob mice pretreated with calpain inhibitor III (25 μ M) or vehicle for 1 h, and then subjected to 4 h of anoxia and 20 min of reoxygenation. Changes in LC3, Beclin-1 and calpain 2 were determined by western blotting at each time points ( e ). Densitometric analysis of PI and TUNEL assay at 20 min of reoxygenation is shown ( f ). # P
    Calpain Cleavage Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/calpain cleavage buffer/product/Millipore
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    calpain cleavage buffer - by Bioz Stars, 2020-05
    90/100 stars
      Buy from Supplier

    Image Search Results


    Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Mouse Assay, Activation Assay

    MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Activity Assay, Expressing, Western Blot

    Calpastatin overexpression inhibits AIF translocation in CA1 neurons after global ischemia. A , Overexpression of calpastatin (Cps) after AAV infection in the rat brain. AAV was infused into the hippocampus using convection methodology to achieve widespread expression in the CA1 sector. Images show HA immunoreactivity (HA–Cps) at 14 d after AAV–Cps infection ( b ); preabsorption of the antibody with Cps protein abolished the signals ( c ). No Cps immunoreactivity is seen in CA1 after empty vector infection ( a ). Western blots (anti-HA or anti-Cps) confirm the expression of Cps protein in the CA1 sector at 7–14 d after AAV–Cps infection; n = 5 per group. B , C , AAV–Cps infection inhibited global ischemia-induced calpain activation in CA1. Western blot ( B ) was performed at 36 h after ischemia to detect α-fodrin cleavage, which was inhibited by AAV–Cps (lanes 6, 7) but not by empty AAV (lanes 4, 5). Substrate-based activity assays ( C ) show that ischemia-induced increases in calpain activity were inhibited by AAV–Cps. * p

    Journal: The Journal of Neuroscience

    Article Title: Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury

    doi: 10.1523/JNEUROSCI.2826-07.2007

    Figure Lengend Snippet: Calpastatin overexpression inhibits AIF translocation in CA1 neurons after global ischemia. A , Overexpression of calpastatin (Cps) after AAV infection in the rat brain. AAV was infused into the hippocampus using convection methodology to achieve widespread expression in the CA1 sector. Images show HA immunoreactivity (HA–Cps) at 14 d after AAV–Cps infection ( b ); preabsorption of the antibody with Cps protein abolished the signals ( c ). No Cps immunoreactivity is seen in CA1 after empty vector infection ( a ). Western blots (anti-HA or anti-Cps) confirm the expression of Cps protein in the CA1 sector at 7–14 d after AAV–Cps infection; n = 5 per group. B , C , AAV–Cps infection inhibited global ischemia-induced calpain activation in CA1. Western blot ( B ) was performed at 36 h after ischemia to detect α-fodrin cleavage, which was inhibited by AAV–Cps (lanes 6, 7) but not by empty AAV (lanes 4, 5). Substrate-based activity assays ( C ) show that ischemia-induced increases in calpain activity were inhibited by AAV–Cps. * p

    Article Snippet: In brief, whole-cell or mitochondrial proteins at the indicated amounts were incubated with calpain reaction buffer [20 m m HEPES, pH 7.6, 1 m m EDTA, 50 m m NaCl, and 0.1% (v/v) 2-mercaptoethanol] containing 10 μ m calpain I fluorescent substrate H-E(EDANS)PLF∼AERK(DABCYL)-OH (Calbiochem, La Jolla, CA).

    Techniques: Over Expression, Translocation Assay, Infection, Convection, Expressing, Plasmid Preparation, Western Blot, Activation Assay, Activity Assay

    Calpain inhibition prevents OGD-induced AIF release in neurons. A , Primary neurons were infected for 3 d with empty AAV or AAV carrying the calpastatin cDNA (AAV–Cps), and the expression of Cps (tagged with triple HA) was confirmed by Western blot using antibodies against Cps and HA, respectively. B , Neurons infected with empty AAV or AAV–Cps were subjected to OGD (60 min), and Western blot was performed to detect α-fodrin cleavage, a marker for calpain activation, at 2 and 6 h after OGD. C , Neurons infected with empty AAV or AAV–Cps were subjected to OGD (60 min), and calpain activity was measured using substrate-based assays at 2, 6, and 16 h after OGD, and the data are expressed as fold increase over non-OGD controls (Con). * p

    Journal: The Journal of Neuroscience

    Article Title: Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury

    doi: 10.1523/JNEUROSCI.2826-07.2007

    Figure Lengend Snippet: Calpain inhibition prevents OGD-induced AIF release in neurons. A , Primary neurons were infected for 3 d with empty AAV or AAV carrying the calpastatin cDNA (AAV–Cps), and the expression of Cps (tagged with triple HA) was confirmed by Western blot using antibodies against Cps and HA, respectively. B , Neurons infected with empty AAV or AAV–Cps were subjected to OGD (60 min), and Western blot was performed to detect α-fodrin cleavage, a marker for calpain activation, at 2 and 6 h after OGD. C , Neurons infected with empty AAV or AAV–Cps were subjected to OGD (60 min), and calpain activity was measured using substrate-based assays at 2, 6, and 16 h after OGD, and the data are expressed as fold increase over non-OGD controls (Con). * p

    Article Snippet: In brief, whole-cell or mitochondrial proteins at the indicated amounts were incubated with calpain reaction buffer [20 m m HEPES, pH 7.6, 1 m m EDTA, 50 m m NaCl, and 0.1% (v/v) 2-mercaptoethanol] containing 10 μ m calpain I fluorescent substrate H-E(EDANS)PLF∼AERK(DABCYL)-OH (Calbiochem, La Jolla, CA).

    Techniques: Inhibition, Infection, Expressing, Western Blot, Marker, Activation Assay, Activity Assay

    Truncation of AIF enables its disassociation with HSP10 in the mitochondria. A , HSP10 is an endogenous binding protein for AIF in neurons. Left, Proteins from cultured neurons were first immunoprecipitated with anti-HSP60 or anti-HSP10 antibody and then immunoblotted with anti-AIF, anti-HSP60, and anti-HSP10 antibodies. Right, Proteins were first immunoprecipitated with anti-AIF or anti-HSP60 antibody and then immunoblotted with anti-AIF, anti-HSP60, and anti-HSP10 antibodies. B , Autoradiograph shows direct protein–protein interaction between AIF and HSP10. The protein-binding assay was performed using in vitro translated proteins (the Flag–HSP10 and [ 35 S]AIF). The reaction products were immunoprecipitated using the anti-Flag antibody, electrophoresed, and then autoradiographed. C , Autoradiograph for [ 35 S]AIF shows that Flag–HSP10 can directly bind to AIF 62 but not to AIF 57 and that calpain I, which cleaves AIF, inhibited AIF 62 /HSP10 binding but not the binding between HSP10 and the calpain-resistant AIFm. The protein-binding assay was performed as described in B . D , Western blots show decreased AIF/HSP10 binding after OGD in empty AAV-infected neurons. Cell extracts were immunoprecipitated using the anti-HSP10 antibody and then immunoblotted with the anti-AIF antibody. The AIF/HSP10 binding was restored in neurons transfected with AAV–Cps. The graph at the bottom illustrates the relative changes of three independent experiments. * p

    Journal: The Journal of Neuroscience

    Article Title: Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury

    doi: 10.1523/JNEUROSCI.2826-07.2007

    Figure Lengend Snippet: Truncation of AIF enables its disassociation with HSP10 in the mitochondria. A , HSP10 is an endogenous binding protein for AIF in neurons. Left, Proteins from cultured neurons were first immunoprecipitated with anti-HSP60 or anti-HSP10 antibody and then immunoblotted with anti-AIF, anti-HSP60, and anti-HSP10 antibodies. Right, Proteins were first immunoprecipitated with anti-AIF or anti-HSP60 antibody and then immunoblotted with anti-AIF, anti-HSP60, and anti-HSP10 antibodies. B , Autoradiograph shows direct protein–protein interaction between AIF and HSP10. The protein-binding assay was performed using in vitro translated proteins (the Flag–HSP10 and [ 35 S]AIF). The reaction products were immunoprecipitated using the anti-Flag antibody, electrophoresed, and then autoradiographed. C , Autoradiograph for [ 35 S]AIF shows that Flag–HSP10 can directly bind to AIF 62 but not to AIF 57 and that calpain I, which cleaves AIF, inhibited AIF 62 /HSP10 binding but not the binding between HSP10 and the calpain-resistant AIFm. The protein-binding assay was performed as described in B . D , Western blots show decreased AIF/HSP10 binding after OGD in empty AAV-infected neurons. Cell extracts were immunoprecipitated using the anti-HSP10 antibody and then immunoblotted with the anti-AIF antibody. The AIF/HSP10 binding was restored in neurons transfected with AAV–Cps. The graph at the bottom illustrates the relative changes of three independent experiments. * p

    Article Snippet: In brief, whole-cell or mitochondrial proteins at the indicated amounts were incubated with calpain reaction buffer [20 m m HEPES, pH 7.6, 1 m m EDTA, 50 m m NaCl, and 0.1% (v/v) 2-mercaptoethanol] containing 10 μ m calpain I fluorescent substrate H-E(EDANS)PLF∼AERK(DABCYL)-OH (Calbiochem, La Jolla, CA).

    Techniques: Binding Assay, Cell Culture, Immunoprecipitation, Autoradiography, Protein Binding, In Vitro, Western Blot, Infection, Transfection

    The wild-type AIF, but not the calpain-resistant mutant AIF, restores OGD-induced cell death of AIF-deficient neurons. A , B , Neurons from Hq mice or wild-type (Wt) mice were infected with AAV–AIF, AAV–AIFm, or empty AAV for 3 d and then subjected to OGD for 60 min. Cell viability ( A ) and cell death ( B ) were measured at 24 and 48 h after OGD using Alamar blue fluorescence and phosphatidylinositol/Hoechst 33258 staining, respectively. * p

    Journal: The Journal of Neuroscience

    Article Title: Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury

    doi: 10.1523/JNEUROSCI.2826-07.2007

    Figure Lengend Snippet: The wild-type AIF, but not the calpain-resistant mutant AIF, restores OGD-induced cell death of AIF-deficient neurons. A , B , Neurons from Hq mice or wild-type (Wt) mice were infected with AAV–AIF, AAV–AIFm, or empty AAV for 3 d and then subjected to OGD for 60 min. Cell viability ( A ) and cell death ( B ) were measured at 24 and 48 h after OGD using Alamar blue fluorescence and phosphatidylinositol/Hoechst 33258 staining, respectively. * p

    Article Snippet: In brief, whole-cell or mitochondrial proteins at the indicated amounts were incubated with calpain reaction buffer [20 m m HEPES, pH 7.6, 1 m m EDTA, 50 m m NaCl, and 0.1% (v/v) 2-mercaptoethanol] containing 10 μ m calpain I fluorescent substrate H-E(EDANS)PLF∼AERK(DABCYL)-OH (Calbiochem, La Jolla, CA).

    Techniques: Mutagenesis, Mouse Assay, Infection, Fluorescence, Staining

    Calpain-dependent truncation of AIF is required for its release after OGD. A , Neurons were infected for 3 d with AAV carrying either the AIF-GFP fusion construct ( a–l ) or the calpain-resistant mutant (L101/103G) AIFm-GFP construct ( m–t ) and then subjected to OGD for 60 min. Confocal images were taken on triple-label immunofluorescent neurons (GFP, green; COX IV, red; Hoechst 33258, blue) in non-OGD cultures ( a–d , m–p ) or at 2 h ( e–h , q–t ) and 6 h ( i–l ) after OGD. In non-OGD neurons, AIF–GFP and AIFm–GFP are localized in mitochondria; after OGD, AIF–GFP is translocated into the nucleus ( e–l ), whereas AIFm–GFP remains in the mitochondria ( q–t ). B , Percentages of transfected neurons showing nuclear localization of AIF–GFP or AIFm–GFP at 2, 6, and 16 h after 60 min OGD. * p

    Journal: The Journal of Neuroscience

    Article Title: Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury

    doi: 10.1523/JNEUROSCI.2826-07.2007

    Figure Lengend Snippet: Calpain-dependent truncation of AIF is required for its release after OGD. A , Neurons were infected for 3 d with AAV carrying either the AIF-GFP fusion construct ( a–l ) or the calpain-resistant mutant (L101/103G) AIFm-GFP construct ( m–t ) and then subjected to OGD for 60 min. Confocal images were taken on triple-label immunofluorescent neurons (GFP, green; COX IV, red; Hoechst 33258, blue) in non-OGD cultures ( a–d , m–p ) or at 2 h ( e–h , q–t ) and 6 h ( i–l ) after OGD. In non-OGD neurons, AIF–GFP and AIFm–GFP are localized in mitochondria; after OGD, AIF–GFP is translocated into the nucleus ( e–l ), whereas AIFm–GFP remains in the mitochondria ( q–t ). B , Percentages of transfected neurons showing nuclear localization of AIF–GFP or AIFm–GFP at 2, 6, and 16 h after 60 min OGD. * p

    Article Snippet: In brief, whole-cell or mitochondrial proteins at the indicated amounts were incubated with calpain reaction buffer [20 m m HEPES, pH 7.6, 1 m m EDTA, 50 m m NaCl, and 0.1% (v/v) 2-mercaptoethanol] containing 10 μ m calpain I fluorescent substrate H-E(EDANS)PLF∼AERK(DABCYL)-OH (Calbiochem, La Jolla, CA).

    Techniques: Infection, Construct, Mutagenesis, Transfection

    Calpain cleavage of GluN2B to isolate Cys cluster II-containing C-terminal fragment reveals reduced palmitoylation in YAC128 striatum. (A) Cartoon indicating relative location of GluN2B Cys clusters I and II and the major calpain cleavage site. Each panel of image data shows results from one representative gel and the dividing line(s) indicate where lanes were removed for ease of comparing genotypes under identical experimental conditions. (B–D) Palmitoylation of Cys cluster II-containing C-terminal fragment was examined by IP/Btn-BMCC method. Representative blots show that palmitoylation (detected by probing with streptavidin Alexa 680, B ) of GluN2B Cys cluster II-containing C-terminal fragment as a ratio to total GluN2B levels (detected by C-terminal specific antibody MA1-2014, as shown in C ) was significantly decreased in striatum from YAC128 compared to FVB/N wild-type mice. Black arrow = ∼62 kDa cluster II-containing C-terminal fragment. (D) Summary graph indicates the corresponding ratio of palmitoylation signal (calculated as in Figure 1 ) for YAC128 normalized to the FVB/N control. Data presented for striatal tissue from 10 independent experiments; bars represent means ± SD ∗ p

    Journal: Frontiers in Synaptic Neuroscience

    Article Title: Altered Regulation of Striatal Neuronal N-Methyl-D-Aspartate Receptor Trafficking by Palmitoylation in Huntington Disease Mouse Model

    doi: 10.3389/fnsyn.2019.00003

    Figure Lengend Snippet: Calpain cleavage of GluN2B to isolate Cys cluster II-containing C-terminal fragment reveals reduced palmitoylation in YAC128 striatum. (A) Cartoon indicating relative location of GluN2B Cys clusters I and II and the major calpain cleavage site. Each panel of image data shows results from one representative gel and the dividing line(s) indicate where lanes were removed for ease of comparing genotypes under identical experimental conditions. (B–D) Palmitoylation of Cys cluster II-containing C-terminal fragment was examined by IP/Btn-BMCC method. Representative blots show that palmitoylation (detected by probing with streptavidin Alexa 680, B ) of GluN2B Cys cluster II-containing C-terminal fragment as a ratio to total GluN2B levels (detected by C-terminal specific antibody MA1-2014, as shown in C ) was significantly decreased in striatum from YAC128 compared to FVB/N wild-type mice. Black arrow = ∼62 kDa cluster II-containing C-terminal fragment. (D) Summary graph indicates the corresponding ratio of palmitoylation signal (calculated as in Figure 1 ) for YAC128 normalized to the FVB/N control. Data presented for striatal tissue from 10 independent experiments; bars represent means ± SD ∗ p

    Article Snippet: In brief, immunoprecipitates from both portions were diluted in 400 μl calpain reaction buffer (20 mM HEPES pH7.6, 10 mM KCl, 1.5 mM MgCl2 , 1 mM dithiothreitol) with 10 nM of recombinant calpain-1 (EMD-Millipore, 208712) and 2 mM CaCl2 for 10 min at RT, except one half was pre-treated with 100 μM of calpain inhibitor CI-III (EMD Millipore, 208722).

    Techniques: Mouse Assay

    Calpain 2 is activated and aggravates I/R injury in fatty livers. ( a ) Lean and ob/ob mice were subjected to 1 h of ischemia and 6 h of reperfusion. Liver calpain 1 and calpain 2 expression levels were analyzed by western blotting. Quantification of the relative protein level is shown (right; n =4 per group). ( b ) Isolated hepatocytes from lean and ob/ob mice underwent 4 h of anoxia and 20 min of reoxygenation. Calpain 1 and calpain 2 expression levels were determined at each time point. Quantification of relative protein level is shown (right; n =4 per group). ( c ) Calpain activity was determined by SLLVY-AMC fluorometry at each time point. ( d ). Ob/ob mice were pretreated with calpain inhibitor III (10 mg/kg) or vehicle DMSO for 6 h and then subjected to 1-h ischemia and 24 h of reperfusion. Necrotic area of liver samples was identified under light microscope (200x). Quantification of the necrotic areas is shown (right; n =4). ( e and f ) Isolated hepatocytes from ob/ob mice pretreated with calpain inhibitor III (25 μ M) or vehicle for 1 h, and then subjected to 4 h of anoxia and 20 min of reoxygenation. Changes in LC3, Beclin-1 and calpain 2 were determined by western blotting at each time points ( e ). Densitometric analysis of PI and TUNEL assay at 20 min of reoxygenation is shown ( f ). # P

    Journal: Cell Death & Disease

    Article Title: Calpain 2-mediated autophagy defect increases susceptibility of fatty livers to ischemia–reperfusion injury

    doi: 10.1038/cddis.2016.66

    Figure Lengend Snippet: Calpain 2 is activated and aggravates I/R injury in fatty livers. ( a ) Lean and ob/ob mice were subjected to 1 h of ischemia and 6 h of reperfusion. Liver calpain 1 and calpain 2 expression levels were analyzed by western blotting. Quantification of the relative protein level is shown (right; n =4 per group). ( b ) Isolated hepatocytes from lean and ob/ob mice underwent 4 h of anoxia and 20 min of reoxygenation. Calpain 1 and calpain 2 expression levels were determined at each time point. Quantification of relative protein level is shown (right; n =4 per group). ( c ) Calpain activity was determined by SLLVY-AMC fluorometry at each time point. ( d ). Ob/ob mice were pretreated with calpain inhibitor III (10 mg/kg) or vehicle DMSO for 6 h and then subjected to 1-h ischemia and 24 h of reperfusion. Necrotic area of liver samples was identified under light microscope (200x). Quantification of the necrotic areas is shown (right; n =4). ( e and f ) Isolated hepatocytes from ob/ob mice pretreated with calpain inhibitor III (25 μ M) or vehicle for 1 h, and then subjected to 4 h of anoxia and 20 min of reoxygenation. Changes in LC3, Beclin-1 and calpain 2 were determined by western blotting at each time points ( e ). Densitometric analysis of PI and TUNEL assay at 20 min of reoxygenation is shown ( f ). # P

    Article Snippet: For in vitro calpain cleavage assays, TNT reaction products were incubated in calpain cleavage buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1 mM DTT) containing 0.3 units/ml recombinant calpain (Calbiochem) and 5 mM CaCl2 .

    Techniques: Mouse Assay, Expressing, Western Blot, Isolation, Activity Assay, Light Microscopy, TUNEL Assay

    Calpain 2 degrades Atg3 and Atg7. ( a ) Lean and ob/ob mice were subjected to 1 h of ischemia and 6 h of reperfusion. The expression of liver autophagy-related proteins Atg3, Atg4B and Atg7 were compared by western blotting (left, n =3). The abundance of Atg3, Atg4B and Atg7 mRNAs were determined by RT-qPCR (right, n =3). ( b and c ) Ob/ob mice were pretreated with calpain inhibitor III (10 mg/kg) or vehicle DMSO for 6 h ( b ), or ob/ob mice were transduced with Ad-shCalpain2 or control virus (Ad-NC; 2 × 10 11 vp/mice) for 7 days, respectively ( c ), and then subjected to 1-h ischemia and 6 h of reperfusion. Liver Atg3, Atg7 and calpain 2 expression levels at each group were determined by western blotting. Quantification of relative protein level is shown in Supplementary Figures 6a and b . ( d ) Hepatocytes isolated from ob/ob mice were subjected to 4 h of anoxia and 20 min of reoxygenation in the presence or absence of calpain inhibitor III (25 μ M) pretreatment for 1 h. Atg3, Atg4B, Atg7 and Calpian 2 expression levels were determined by western blotting at indicated time points. ( e ) Hepatocytes isolated from ob/ob mice infected with Ad-shCalpain2 or control virus (Ad-NC) for 48 h and then subjected to 4 h of anoxia and 20 min of reoxygenation. Western blotting of the relative protein level is shown. Quantification of the relative protein level of ( d ) and ( e ) is shown in Supplementary Figures 6c and d . ( f and g ) For the in vitro calpain cleavage assay, wild-type Atg7 or Atg7 mutant at the 344–349 amino acid ( f ), Atg3 or Atg3 mutant at the 92–97 amino acid ( g ) recombinant proteins that translated by TnT Translation System, were incubated with recombinant calpain in the presence or absence of 25 μ M calpain inhibitor III at 30 °C for 10 min. Then the reaction was terminated and subjected to western blot. * P

    Journal: Cell Death & Disease

    Article Title: Calpain 2-mediated autophagy defect increases susceptibility of fatty livers to ischemia–reperfusion injury

    doi: 10.1038/cddis.2016.66

    Figure Lengend Snippet: Calpain 2 degrades Atg3 and Atg7. ( a ) Lean and ob/ob mice were subjected to 1 h of ischemia and 6 h of reperfusion. The expression of liver autophagy-related proteins Atg3, Atg4B and Atg7 were compared by western blotting (left, n =3). The abundance of Atg3, Atg4B and Atg7 mRNAs were determined by RT-qPCR (right, n =3). ( b and c ) Ob/ob mice were pretreated with calpain inhibitor III (10 mg/kg) or vehicle DMSO for 6 h ( b ), or ob/ob mice were transduced with Ad-shCalpain2 or control virus (Ad-NC; 2 × 10 11 vp/mice) for 7 days, respectively ( c ), and then subjected to 1-h ischemia and 6 h of reperfusion. Liver Atg3, Atg7 and calpain 2 expression levels at each group were determined by western blotting. Quantification of relative protein level is shown in Supplementary Figures 6a and b . ( d ) Hepatocytes isolated from ob/ob mice were subjected to 4 h of anoxia and 20 min of reoxygenation in the presence or absence of calpain inhibitor III (25 μ M) pretreatment for 1 h. Atg3, Atg4B, Atg7 and Calpian 2 expression levels were determined by western blotting at indicated time points. ( e ) Hepatocytes isolated from ob/ob mice infected with Ad-shCalpain2 or control virus (Ad-NC) for 48 h and then subjected to 4 h of anoxia and 20 min of reoxygenation. Western blotting of the relative protein level is shown. Quantification of the relative protein level of ( d ) and ( e ) is shown in Supplementary Figures 6c and d . ( f and g ) For the in vitro calpain cleavage assay, wild-type Atg7 or Atg7 mutant at the 344–349 amino acid ( f ), Atg3 or Atg3 mutant at the 92–97 amino acid ( g ) recombinant proteins that translated by TnT Translation System, were incubated with recombinant calpain in the presence or absence of 25 μ M calpain inhibitor III at 30 °C for 10 min. Then the reaction was terminated and subjected to western blot. * P

    Article Snippet: For in vitro calpain cleavage assays, TNT reaction products were incubated in calpain cleavage buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1 mM DTT) containing 0.3 units/ml recombinant calpain (Calbiochem) and 5 mM CaCl2 .

    Techniques: Mouse Assay, Expressing, Western Blot, Quantitative RT-PCR, Transduction, Isolation, Infection, In Vitro, Cleavage Assay, Mutagenesis, Recombinant, Incubation