Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: Only enzymatically active FKBP38 interacts with Bcl-2. (A) Co-immunoprecipitation of endogenous FKBP38 and Bcl-2. After preincubation with 500 μM EGTA, SH-SY5Y cell lysate was incubated with rabbit anti-FKBP38 antibody. Antibody/protein complexes were bound to protein G-Sepharose. Samples were washed. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using mouse anti-Bcl-2 antibody. Cell lysate preincubated with rabbit immunoglobin was used as a control. FKBP38 bound to Bcl-2 in the presence of 1 mM calcium. This interaction was disrupted by 1 μM GPI1046. No interaction was observed in the absence of Ca2+. (B) SH-SY5Y crude cell extract was applied in the absence and presence of 20 μM CaM and 2 mM Ca2+ to maltose-binding protein-Bcl-2 fusion protein (MBP-Bcl-2) immobilized on amylose resin. After three washing steps, protein was eluted by 200 mM maltose and analyzed by Western blot using polyclonal anti-FKBP38 antibody. The proportion of active endogenous FKBP38 was quantified by Biorad Multi-Analyst software. (C) MBP-Bcl-2 was immobilized on amylose resin and incubated with (1) FKBP38, (2) FKBP38 and CaM, (3) FKBP38 and Ca2+/CaM and (4) FKBP38, Ca2+/CaM and 200 nM GPI1046. After washing, protein was eluted and analyzed by Western blot using polyclonal anti-FKBP38 antibody. (D) Inhibition of PPIase activity of 1 μM FKBP38 by Bcl-2 was measured in the PPIase assay in the presence of 5 mM CaCl2 and 5 μM CaM. The calculated Ki value was 0.74 μM.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot, Binding Assay, Software, Inhibition, Activity Assay

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: FKBP38 activity influences Bcl-2 function. (A) Near-UV CD spectra of 1 μM Ca2+/CaM/FKBP38 and 3 μM Bcl-2 were measured either separated (dotted line) or mixed (solid line) in a tandem cuvette. (B) Co-immunoprecipitation. SH-SY5Y cells were stimulated to apoptosis by 50 μM etoposide. Cell lysate preincubated with 500 μM EGTA was incubated with monoclonal mouse anti-Bcl-2 antibody. Antibody/protein complexes were bound to protein G-Sepharose. Samples were washed. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using rabbit anti-FKBP38 antibody. Cell lysate preincubated with control mouse immunoglobin was used as a control. FKBP38 bound to Bcl-2 in the presence of 1 mM Ca2+, whereas 1 μM GPI1046 abolished the interaction. Bcl-2/Bad interaction was investigated by incubation of SH-SY5Y cell lysate with hamster anti-Bcl-2 antibody and protein was detected using mouse anti-Bad antibody. Bad/Bcl-2 interaction was observed in the presence of 1 mM calcium and 1 μM GPI1046. In addition, SH-SY5Y cell lysate was incubated with monoclonal mouse anti-Bad antibody, and antibody/protein complexes formed were bound to protein G-Sepharose. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using hamster anti-Bcl-2 antibody. Cell lysate preincubated with mouse immunoglobin was used as a control. Bcl-2 bound to Bad in the presence of 1 mM Ca2+ and 1 μM GPI1046, indicating that GPI1046 interfered with the Bcl-2/FKBP38 interaction, allowing Bad/Bcl-2 complexes to form. (C) MBP-Bcl-2 fusion protein was immobilized on amylose beads and incubated with Bad (Sigma), FKBP38 and CaM either in the presence or absence of 2 mM Ca2+. Pellet (P) and supernatant (S) were subjected to 12.5% SDS–PAGE and analyzed by Western blotting using mouse anti-Bad antibody and hamster anti-Bcl-2 antibody. (D) Subcellular distribution of Bcl-2 and Bad in SH-SY5Y neuroblastoma cells was analyzed by immunostaining with FITC-conjugated goat anti-mouse antibody against mouse anti-Bad antibody and Cy5-conjugated goat anti-hamster antibody against hamster anti-Bcl-2 antibody. Nuclei were stained with DAPI. Cells were treated for 16 h with 50 μM etoposide either in the absence or presence of 1 μM GPI1046. Cells transfected with FKBP38 RNAi construct were analyzed with ImageBlue-conjugated goat anti-mouse antibody against mouse anti-Bad antibody and TRITC-conjugated goat anti-hamster antibody against hamster anti-Bcl-2. Nuclei were stained by 7-AAD. Because of better understanding, colors of this panel were adapted to other results presented in this figure. Again, inhibition of FKBP38 activity allows Bcl-2/Bad interaction after induction of apoptosis by 50 μM etoposide.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Activity Assay, Immunoprecipitation, Incubation, SDS Page, Western Blot, Immunostaining, Staining, Transfection, Construct, Inhibition

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: FKBP38 and Bcl-2 colocalize in neuroblastoma cells. (A) Localization of Bcl-2 and FKBP38 in SH-SY5Y neuroblastoma cells was analyzed by immunostaining with Cy5-conjugated goat anti-hamster antibody against hamster anti-Bcl-2 antibody and FITC-conjugated goat anti-rabbit IgG against rabbit anti-FKBP38 antibody. Cells were treated for 16 h with 50 μM etoposide and 2 μM GPI1046. Nuclei were stained with DAPI. (B) Subcellular distribution of FKBP38 in SH-SY5Y cells was studied by immunostaining with FITC-conjugated goat anti-rabbit IgG against rabbit anti-FKBP38 antibody and a subcellular structure localization kit (Chemicon).

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Immunostaining, Staining

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: (C) Subcellular localization of FKBP38 and Bcl-2 was studied by preparing mitochondria, ER, nucleus and cytosol from SH-SY5Y cells and analyzing these fractions by Western blotting using anti-FKBP38 and anti-Bcl-2 antibodies. Antibodies detecting cytochrome c (Cyt c) and FKBP13 were used as controls for mitochondrial and ER localization, respectively.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Western Blot

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Quantitative real-time PCR of miR-145 expression in glioma samples (n = 19) compared with normal samples (n = 10). (B) quantitative real-time PCR of miR-145 expression in rat glioma tissues (n = 6) and U87, U251 glioma cells compared to normal tissues (n = 6). (C) Quantitative real-time PCR of BNIP3 mRNA expression in glioma samples (n = 19) compared with normal samples (n = 10). (D) quantitative real-time PCR of BNIP3 mRNA expression in rat glioma tissues and U87, U251 glioma cells compared to normal tissues. (E) Pathology observation of mice brain tissues sections stained with IHC (×100, ×200, ×400). (F) Immunofluorescence with BNIP3 (green) in rat normal tissues and glioma tissues (×200).*p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Immunofluorescence, Control

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Western analysis of BNIP3 and control β-actin in glioma samples compared with normal samples. (B) Western analysis of BNIP3 and control β-actin in rat glioma tissues compared to normal tissues. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Western Blot, Control

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Bioinformatics analysis shows the seed sequence of miR-145 binding to the 3′-UTR of BNIP3 mRNA. (B) , (C) Quantitative real-time PCR analysis of mRNA expression of BNIP3 in U87 cells treated with miR-145 mimics and inhibitor for 48 h. (D) Western analysis of protein expression of BNIP3 in U87 and U251 cells treated with miR-145 mimics and inhibitor for 48 h. (E) Immunofluorescence with BNIP3 (green) in U87 and U251 cells after miR-145 mimics or mimics NC treatment.*p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Sequencing, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Control

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Western analysis of BNIP3, which is localized in the nucleus or the cytoplasm in U87 and U251 cells treated with miR-145 mimics and inhibitor for 48 h. (B) Wild-type 3′-UTR of BNIP3 gene was cloned into the firefly and Renilla reporter plasmid. The BNIP3-3′UTR constructs or blank plasmid were transfected into U87 and U251 cells with control or miR-145 mimics, followed by dual luciferase assays. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Western Blot, Clone Assay, Plasmid Preparation, Construct, Transfection, Control, Luciferase

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A, B) Transfection effect of BNIP3-siRNA or BNIP3-vector was confirmed by quantitative real-time PCR. (C) U87 cells and U251 cells were stained with Hoechst 33342 dye after BNIP3-siRNA or control treatment. (D) U87 and U251 cells were stained with Tunel after BNIP3-siRNA or control treatment. (E) U87 cell apoptosis after BNIP3-siRNA or control treatment was determined by FACS. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Staining, Control, TUNEL Assay

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A, B) Protein expression of Notch1, p21 and Hes1 was determined by western blot analysis in U87 and U251 cells transfected with miR-145 mimics and mimics-NC, or miR-145 inhibitor and inhibitor-NC. (C, D) Western analysis of Notch1-related proteins in U87 and U251 cells transfected with Bnip3 siRNA and siRNA control, or BNIP3 expression vector and blank vector. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Protein expression of Notch1, p21, Hes1 was determined by western blot analysis in U87 and U251 cells co-transfected with miR-145 inhibitor and BNIP3-siRNA, or with miR-145 inhibitor. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Expressing, Western Blot, Transfection, Control

Journal: The Journal of Clinical Investigation

Article Title: Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

doi: 10.1172/JCI76344

Figure Lengend Snippet: (A) Immunoblot for beclin 1 using an antibody that recognizes aa 171–291 of the protein. Blots are representative of Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl (n = 4) mice. (B) Immunoblot for beclin 1 in Hmgb1fl/fl Il10–/– (n = 4) and Vil-Cre Hmgb1fl/fl Il10–/– (n = 4) mice. (C) Beclin 1 immunoblot of the products from an in vitro cleavage assay of beclin 1 by calpain 1 with decreasing amounts of HMGB1. (D) MYC-DDK (FLAG) immunoblot of the products of the beclin 1 in vitro cleavage assay. Recombinant beclin 1 contained a C-terminal MYC-DDK epitope tag. (E) Immunoblot for ATG5 using an antibody that recognizes aa 2–15 of the protein. Blots are representative of Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl (n = 4) mice. (F) Immunoblot for ATG5 in Hmgb1fl/fl Il10–/– (n = 4) and Vil-Cre Hmgb1fl/fl Il10–/– (n = 4) mice. (G) ATG5 immunoblot of the products from an in vitro cleavage assay of ATG5 by calpain 1 with decreasing amounts of HMGB1. (H) Co-IP of HMGB1 and beclin 1 or ATG5 in colonic mucosal lysates from Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl (n = 4) mice treated with DSS for 3 days. (I) Calpain activity assay evaluating cleavage of a fluorogenic calpain 1/2 substrate (Suc-LLVY-AMC) in samples of colonic mucosa from Hmgb1fl/fl (n = 3) and Vil-Cre Hmgb1fl/fl mice (n = 3) on day 3 of DSS treatment (mean ± SEM). (J) Immunoblot for the active p20 fragment of caspase 1 in Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl mice (n = 4) on day 3 of DSS treatment. (K) qRT-PCR for calpastatin in cDNA from Hmgb1fl/fl (n = 6) and Vil-Cre Hmgb1fl/fl mice (n = 6) on day 3 of DSS treatment (mean ± SEM). Data were analyzed by 2-tailed Student’s t tests. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. RFU, relative fluorescence units.

Article Snippet: For coimmunoprecipitation (Co-IP) of HMGB1 and beclin 1, anti-HMGB1 (Ab18256; Abcam) and anti–beclin 1 (612113; BD Biosciences) were used.

Techniques: Western Blot, In Vitro, Cleavage Assay, Recombinant, Co-Immunoprecipitation Assay, Activity Assay, Quantitative RT-PCR, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

doi: 10.1172/JCI76344

Figure Lengend Snippet: Hmgb1fl/fl and Vil-Cre Hmgb1fl/fl mice were administered 2.5% DSS in their drinking water for 5 days and then sacrificed. Vil-Cre Hmgb1fl/fl mice were also treated with vehicle control or the calpain inhibitor calpeptin (5 mg/kg) by daily i.p. injection. (A) Weight loss on day 5 of DSS administration (mean). (B) H&E-stained sections of formalin-fixed, paraffin-embedded intestines from Vil-Cre Hmgb1fl/fl mice treated with vehicle (n = 4) or calpeptin (n = 4). Images were from areas 10 mm proximal to the animal’s rectum (original magnification, ×400). (C) Immunoblot for beclin 1 in intestinal mucosal scrapings from untreated Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl mice treated with vehicle (n = 4) or calpeptin (n = 4). (D) Immunoblot for ATG5 in intestinal mucosal scrapings from untreated Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl mice treated with vehicle (n = 4) or calpeptin (n = 4). Data were analyzed by 1-way ANOVA with Bonferroni’s multiple comparisons test. **P < 0.01.

Article Snippet: For coimmunoprecipitation (Co-IP) of HMGB1 and beclin 1, anti-HMGB1 (Ab18256; Abcam) and anti–beclin 1 (612113; BD Biosciences) were used.

Techniques: Injection, Staining, Formalin-fixed Paraffin-Embedded, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

doi: 10.1172/JCI76344

Figure Lengend Snippet: IEC progenitors were isolated from the small intestines of Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl (n = 4) mice and grown in culture until intestinal enteroids formed. The cells were then treated with 10 μg/ml L-18 MDP and lysed in 1% Triton lysis buffer. (A) Immunoblot for HMGB1 in lysates from cells treated with MDP for 4 hours. (B) Immunoblot for beclin 1 in lysates from cells treated with MDP for 4 hours. (C) Immunoblot for active caspase 3 in lysates from cells treated with MDP for 4 hours. (D) Immunoblot for active caspase 3 in lysates from cells treated with MDP for 4 hours in the presence of DMSO (vehicle control) or 1 μg/ml calpeptin. (E) Immunoblot for LC3B in lysates treated with MDP for 4 hours in the presence or absence of 100 nM bafilomycin A1. (F) Immunoblot for beclin 1 in lysates from cells treated with MDP at the indicated time points. (G) Immunoblot for ATG5 in lysates from cells treated with MDP at the indicated time points. (H) Immunoblot for cleaved caspase 3 in lysates from cells treated with MDP at the indicated time points. (I) Calpain activity in enteroid lysates at the indicated times after MDP treatment. Data were analyzed by 2-way ANOVA with Bonferroni’s multiple comparisons test as well as the 2-tailed Student’s t test for between-genotype comparisons within a treatment group **P < 0.01; ****P < 0.001.

Article Snippet: For coimmunoprecipitation (Co-IP) of HMGB1 and beclin 1, anti-HMGB1 (Ab18256; Abcam) and anti–beclin 1 (612113; BD Biosciences) were used.

Techniques: Isolation, Lysis, Western Blot, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

doi: 10.1172/JCI76344

Figure Lengend Snippet: (A) HMGB1 expression by qRT-PCR in controls (n = 6) and in patients with active UC (n = 5) or indeterminate colitis (IC) (n = 1) (mean ± SEM). Immunoblot for HMGB1 protein in lysates of snap-frozen endoscopic biopsies. Lanes 1–4, controlled CD patients; lanes 5–8, active CD patients (mean ± SEM). (B) Confocal microscopic images of endoscopic biopsies stained for Hoechst (blue) and HMGB1 (red) in controls (n = 3) and in patients with active UC (n = 4) (original magnification, ×400). Colocalization evaluated using Pearson’s correlation coefficient with the Costes correction (mean ± SEM). (C) Beclin 1 immunoblot. Lane 1, control/normal; lane 2, control/normal; lane 3, control/quiescent CD; lane 4, moderate UC; lane 5, moderate UC; and lane 6, severe CD (mean ± SEM). (D) Immunoblot for ATG5 in lysates of snap-frozen endoscopic biopsies (mean ± SEM). Samples were loaded as in A. (E) Immunoblot with antibody recognizing the active p19/p17 fragments of cleaved caspase 3. Samples were loaded as in Figure 4D. (F) Immunoblot for the active p20 fragment of cleaved caspase 1. Samples were loaded as in Figure 4D. (G) Calpain activity as evaluated by cleavage of the fluorescent substrate in samples from controls (n = 3) and from patients with active UC (n = 3) (mean ± SEM). (H) Calpastatin levels analyzed by qRT-PCR in controls (n = 6) and in patients with active UC (n = 5) or IC (n = 1) (mean ± SEM). Data were analyzed by 2-tailed Student’s t tests, except for data in G, which were determined by the 1-tailed Student’s t test. *P < 0.05; **P < 0.01; ****P < 0.001.

Article Snippet: For coimmunoprecipitation (Co-IP) of HMGB1 and beclin 1, anti-HMGB1 (Ab18256; Abcam) and anti–beclin 1 (612113; BD Biosciences) were used.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

doi: 10.1172/JCI76344

Figure Lengend Snippet: (A) Calpain activation occurs secondarily to inflammasome-mediated caspase 1 activation with subsequent degradation and downregulation of the calpain inhibitor calpastatin. Low levels of calpastatin in the presence of calcium lead to autoactivation of calpains. Inflammasome-mediated caspase 1 activation is terminated by autophagic degradation of inflammasomes. (B) HMGB1 interacts with beclin 1 and ATG5 to prevent calpain-mediated cleavage of these proteins, allowing autophagy to proceed. (C) In the absence of HMGB1, beclin 1 and ATG5 are cleaved by calpain, generating protein fragments that localize to the mitochondria and trigger cell death.

Article Snippet: For coimmunoprecipitation (Co-IP) of HMGB1 and beclin 1, anti-HMGB1 (Ab18256; Abcam) and anti–beclin 1 (612113; BD Biosciences) were used.

Techniques: Activation Assay