Journal: PLoS ONE

Article Title: Glutamine Treatment Attenuates Endoplasmic Reticulum Stress and Apoptosis in TNBS-Induced Colitis

doi: 10.1371/journal.pone.0050407

Figure Lengend Snippet: (A–E). Protein from colonic extracts was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting for CHOP, BiP, calpain-1 and caspase-12. CHOP, BiP, calpain-1 and caspase-12 were markedly expressed in rats treated with TNBS alone. However, glutamine administration partially abolished CHOP, BiP, calpain-1 and caspase-12 expression induced by TNBS. Results are representative of four independent experiments. Equal loading of proteins is illustrated by β-actin bands. (A) Representative Western-blot photographs for CHOP, calpain-1, BiP, caspase-12, and β-actin. (B) Densitometric quantification of CHOP. (C) Densitometric quantification of calpain-1. (D) Densitometric quantification of BiP. (E) Densitometric quantification of caspase-12. Data are expressed as mean ± S.E.M. from 8 rats. *P<0.05 compared with control group. # P<0.05 compared with TNBS group. & P<0.05 compared with same group 2 d. (F) Photomicrographs of immunohistochemistry for BiP in sections of colonic samples. Paraffin-embedded sections were immunostained with a BiP antibody. Original magnification: 200X.

Article Snippet: The membranes were then blocked with 5% non-fat dry milk in phosphate buffered saline buffer containing 0.05% Tween 20 (PBST) for 1 hour at room temperature and probed overnight at 4°C with polyclonal anti-Bax, Bcl-2, Bcl-xL, poly(ADP-ribose) polymerase-1 (PARP-1), cytochrome c, transcription factor CHOP/GADD153 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), JNK, phospho-JNK, BiP/glucose-regulated protein 78 (GRP78), p53, phospho-p53, cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), calpain-1, caspase-12 and phospho-IRE1 (Abcam, Cambridge, UK) antibodies at 1∶200–1∶1,000 dilution with PBST containing 3% non-fat dry milk.

Techniques: Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Immunohistochemistry

Journal: PLoS ONE

Article Title: Combination Therapy with Gossypol Reveals Synergism against Gemcitabine Resistance in Cancer Cells with High BCL-2 Expression

doi: 10.1371/journal.pone.0050786

Figure Lengend Snippet: Immunoblot of anti-apoptotic (pAKT and PI3KR2) and pro-apoptotic (BAD, CASP6 and CAPN1) genes in GEM-R cell line and GEM-S cell lines. Cells were treated with gemcitabine and combination with gossypol for 48 hours. The results shown are representative of two independent experiments.

Article Snippet: The respective primary antibodies were used as follows: anti-Bcl-xl (Cell Signaling; MA, US) at 1∶1000 dilution, anti-Bad (Cell Signaling; MA, US) at 1;1000 dilution, anti-Bak (Cell Signaling; MA, US) 1∶1000 dilution, anti-Bax (Cell Signaling; MA, US) 1∶3000 dilution, anti-caspase-6 (Cell Signaling; MA, US) 1∶1000 dilution, anti-P1K3R2 (Cell Signaling; MA, US) 1∶1000 dilution, anti-pAKT (Cell Signaling; MA, US) at 1∶1000 dilution, anti-calpain-1 (Cell Signaling; MA, US) at 1∶1000 dilution, anti-Actin (loading control; Cell Signaling; MA, US) at 1∶3000 dilution, anti-Bcl-2 (Santa Cruz Biotechnology; CA, USA) 1∶3000 dilution, and anti-Mcl-1 (Santa Cruz Biotechnology; CA, USA) at 1∶3000 dilution, and anti-Noxa (Calbiochem, Merck; Germany) 1∶1000 dilution.

Techniques: Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress

doi: 10.3389/fcell.2022.1008542

Figure Lengend Snippet: Vimentin cleavage is blocked by inhibitors of the proteasome and calpains, which are elevated in GAN. (A) Immunoblotting for vimentin (top) and actin (bottom; loading control) in Triton X insoluble pellet fractions from untreated (lanes 1–3, 7–9) or water-exposed (lanes 4–6, 10–12) control 56.3 and GAN 56.1 fibroblasts in the absence or presence of the DMSO vehicle control, calpain inhibitor (MDL-28170), or calpain/proteasome inhibitor (MG-132). Note partial cleavage inhibition by MDL and near-complete inhibition by MG-132. (B) Immunoblotting for vimentin (top) and actin (bottom; loading control) in reduced Triton X insoluble fractions and for calpain-1 (middle top), calpain-2 (middle), and calpastatin (middle bottom) in reduced Triton X soluble fractions from GAN and control fibroblasts exposed to 8 min of hypotonic stress and treated with the DMSO vehicle control or MG-132. Shown are technical replicates from an individual experiment. The experiment was independently repeated at least 3 times. (C) Quantification of the relative fragment 1 (FR1) and fragment 2 (FR2) band intensities normalized to the full length vimentin band for each cell line (GAN 56.1 fibroblasts; control 56.3 fibroblasts) and treatment group (from panel B). **** p < 0.0001; two-way ANOVA. (D) Quantification of calpain-1, calpain-2, and calpastatin levels (from panel B). ** p < 0.01; **** p < 0.0001; two-way ANOVA.

Article Snippet: The following primary antibodies and concentrations were utilized: rabbit anti-Vimentin (Cell Signaling Technology, D21H3, WB 1:1,000, IF 1:200), mouse anti-Vimentin (Invitrogen, V9, WB 1:1,000–1:2,500, IF 1:100), mouse anti-Actin (Thermo Fisher Scientific, ACTN05, WB 1:1,000), mouse anti-Actin (Santa Cruz, SPM161, WB 1:1,000), rabbit anti-Calpastatin (Cell Signaling Technology, WB 1:1,000), rabbit anti-Calpain 1 (Cell Signaling Technology, large subunit Mu-type, WB 1:1,000), rabbit anti-Calpain 2 (Cell Signaling Technology, large subunit M-type, WB 1:1,000), and rabbit anti-Calpain 2 (Cell Signaling Technology, large subunit M-type, E3M6E, IF 1:200).

Techniques: Western Blot, Inhibition

Journal: Frontiers in Medicine

Article Title: Calpain Inhibitor Calpeptin Alleviates Ischemia/Reperfusion-Induced Acute Kidney Injury via Suppressing AIM2 Inflammasome and Upregulating Klotho Protein

doi: 10.3389/fmed.2022.811980

Figure Lengend Snippet: Calpain 1 and 2 are markedly increased in the renal biopsy of AKI patients. Representative immunohistochemical micrographs of Calpain 1 (A) and Calpain 2 (C) protein expression in the renal biopsy of both AKI patients ( n = 10) and normal control group ( n = 6). Quantitative analysis of Calpain 1 (B) and Calpain 2 (D) expression in the human renal specimens. ×200, bar = 100 μm. Data were presented as mean ± SEM ( n = 3). ** p < 0.01; *** p < 0.001 vs. normal control group. AKI, acute kidney injury.

Article Snippet: The membrane was blocked by protein free rapid blocking buffer (PS108P, Epizyme) for 20 min and then incubated overnight with primary antibodies against Calpain 1 (1:1000, #2556, Cell Signaling Technology), Calpain 2 (1:1000, # DF7807, Affinity), Klotho (1:500, # DF10309, Affinity), AIM2(1:500, # DF3514, Affinity), NLRP3 (1:500, # DF7438, Affinity), pro-Caspase 1 (1:500, #24232, Cell Signaling Technology), cleaved-Caspase 1 (1:500, #89332, Cell Signaling Technology), ASC (1:1000, sc-514414, Santa Cruz), IL-1β (1:500, #83186, Cell Signaling Technology), IL-18 (1:500, # DF6252, Affinity), LCN2 (1:500, #DF6816, Affinity), and GAPDH (1:10000, ab181602, Abcam).

Techniques: Immunohistochemical staining, Expressing

Journal: Frontiers in Medicine

Article Title: Calpain Inhibitor Calpeptin Alleviates Ischemia/Reperfusion-Induced Acute Kidney Injury via Suppressing AIM2 Inflammasome and Upregulating Klotho Protein

doi: 10.3389/fmed.2022.811980

Figure Lengend Snippet: Calpeptin inhibits AIM2 and NLRP3 inflammasome-mediated inflammation, and upregulates Klotho protein expression in IR mouse model. (A) Western blotting of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, pro-Caspase 1, cleaved-Caspase 1, IL-1β and IL-18 in the kidney of all mice. (B) Quantitative determination of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, cleaved-Caspase 1 and IL-18. (C) The mRNA levels of Calpain 2, Klotho, AIM2, ASC and GSDMD in the kidney among different groups. (D) The calpain activity of renal issues was measured by the relative fluorescence units (400/505 nm). (E) The cathepsin B activity of kidney issues was tested by the relative fluorescence units (400/505 nm). (F) Representative immunohistochemical micrographs from the kidney issues of different groups stained with Calpain 1, Calpain 2 and AIM2. ×400, bar = 50 μm. All data were presented as mean ± SEM ( n = 3). NS, no significance; * p < 0.05 vs. sham group; ** p < 0.01 vs. sham group; *** p < 0.001 vs. sham group; # p < 0.05 vs. IR group; ## p < 0.01 vs. IR group; ### p < 0.001 vs. IR group. CP, calpeptin; IR, ischemia/reperfusion; RFU, relative fluorescence units.

Article Snippet: The membrane was blocked by protein free rapid blocking buffer (PS108P, Epizyme) for 20 min and then incubated overnight with primary antibodies against Calpain 1 (1:1000, #2556, Cell Signaling Technology), Calpain 2 (1:1000, # DF7807, Affinity), Klotho (1:500, # DF10309, Affinity), AIM2(1:500, # DF3514, Affinity), NLRP3 (1:500, # DF7438, Affinity), pro-Caspase 1 (1:500, #24232, Cell Signaling Technology), cleaved-Caspase 1 (1:500, #89332, Cell Signaling Technology), ASC (1:1000, sc-514414, Santa Cruz), IL-1β (1:500, #83186, Cell Signaling Technology), IL-18 (1:500, # DF6252, Affinity), LCN2 (1:500, #DF6816, Affinity), and GAPDH (1:10000, ab181602, Abcam).

Techniques: Expressing, Western Blot, Activity Assay, Fluorescence, Immunohistochemical staining, Staining

Journal: Frontiers in Medicine

Article Title: Calpain Inhibitor Calpeptin Alleviates Ischemia/Reperfusion-Induced Acute Kidney Injury via Suppressing AIM2 Inflammasome and Upregulating Klotho Protein

doi: 10.3389/fmed.2022.811980

Figure Lengend Snippet: Calpeptin inhibits Calpain 1 activation and GSDMD cleavage in the tubules of IR mice, reduces cells apoptosis as well. (A,B) Representative immunofluorescence micrographs from mice kidneys of different groups stained Calpain 1 and GSDMD. ×400, bar = 50 μm. (C) Representative TUNEL staining micrographs in the mice kidneys of different groups. ×400, bar = 50 μm. (D) Quantitative determination of apoptosis cells amount. All Values were presented as mean ± SEM ( n = 3). *** p < 0.001 vs. sham group; ### p < 0.001 vs. IR group. CP, calpeptin; IR, ischemia/reperfusion.

Article Snippet: The membrane was blocked by protein free rapid blocking buffer (PS108P, Epizyme) for 20 min and then incubated overnight with primary antibodies against Calpain 1 (1:1000, #2556, Cell Signaling Technology), Calpain 2 (1:1000, # DF7807, Affinity), Klotho (1:500, # DF10309, Affinity), AIM2(1:500, # DF3514, Affinity), NLRP3 (1:500, # DF7438, Affinity), pro-Caspase 1 (1:500, #24232, Cell Signaling Technology), cleaved-Caspase 1 (1:500, #89332, Cell Signaling Technology), ASC (1:1000, sc-514414, Santa Cruz), IL-1β (1:500, #83186, Cell Signaling Technology), IL-18 (1:500, # DF6252, Affinity), LCN2 (1:500, #DF6816, Affinity), and GAPDH (1:10000, ab181602, Abcam).

Techniques: Activation Assay, Immunofluorescence, Staining, TUNEL Assay

Journal: Frontiers in Medicine

Article Title: Calpain Inhibitor Calpeptin Alleviates Ischemia/Reperfusion-Induced Acute Kidney Injury via Suppressing AIM2 Inflammasome and Upregulating Klotho Protein

doi: 10.3389/fmed.2022.811980

Figure Lengend Snippet: Calpeptin suppresses AIM2 and NLRP3 inflammasome signaling pathway, and increases Klotho protein expression in CoCl 2 -induced HK-2 cells hypoxia model. (A) Western blotting of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, pro-Caspase 1, cleaved-Caspase 1, ASC, IL-1β and LCN2 in the HK-2 cells. (B) Quantitative determination of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, cleaved-Caspase 1, IL-1β and LCN2. (C) The calpain activity of HK-2 cells was assessed by the relative fluorescence units (400/505 nm). (D) The cathepsin B activity of HK-2 cells was analyzed by the relative fluorescence units (400/505 nm). All data were presented as mean ± SEM ( n = 3). NS, no significance; * p < 0.05 vs. control group; ** p < 0.01 vs. control group; *** p < 0.001 vs. control group; # p < 0.05 vs. CoCl 2 group; ## p < 0.01 vs. CoCl 2 group; ### p < 0.001 vs. CoCl 2 group. CP, calpeptin; RFU, relative fluorescence units.

Article Snippet: The membrane was blocked by protein free rapid blocking buffer (PS108P, Epizyme) for 20 min and then incubated overnight with primary antibodies against Calpain 1 (1:1000, #2556, Cell Signaling Technology), Calpain 2 (1:1000, # DF7807, Affinity), Klotho (1:500, # DF10309, Affinity), AIM2(1:500, # DF3514, Affinity), NLRP3 (1:500, # DF7438, Affinity), pro-Caspase 1 (1:500, #24232, Cell Signaling Technology), cleaved-Caspase 1 (1:500, #89332, Cell Signaling Technology), ASC (1:1000, sc-514414, Santa Cruz), IL-1β (1:500, #83186, Cell Signaling Technology), IL-18 (1:500, # DF6252, Affinity), LCN2 (1:500, #DF6816, Affinity), and GAPDH (1:10000, ab181602, Abcam).

Techniques: Expressing, Western Blot, Activity Assay, Fluorescence