rabbit polyclonal anti cleaved caspase3  (Proteintech)

Journal: The Journal of Clinical Investigation

Article Title: Accumulation of α -synuclein mediates podocyte injury in Fabry nephropathy

doi: 10.1172/JCI157782

Figure Lengend Snippet: ( A ) Transmission electron microscopy (TEM) comparison of foot processes between control and Fabry kidney biopsy before and after ERT with many foot processes widened in Fabry biopsies both untreated and after ERT. Asterisks show Gb3 inclusions in podocytes. Original magnification, ×52,800. ( B ) Significant decrease of podocyte Gb3 inclusions after ERT but persistence of increased foot process width. ( C ) Schematic overview of GLA -knockout (KO) podocyte generation by CRISPR/Cas9 genome editing. ( D ) Western blots show a complete absence of GLA expression in several GLA -KO clones. ( E ) Abolished GLA activity in 2 KO clones compared with WT cells. ( F ) Mass spectrometry analysis confirms the accumulation of Gb3-C24-0 isoform in KO cells, normalized upon 96 hours of α-galactosidase therapy ( n = 3). ( G ) TEM shows zebra bodies exclusively in GLA -KO clones (red arrowheads). While WT cells depict a normal ultrastructure, aGAL-treated KO cells have remnant vacuoles (green arrowheads) without zebra bodies. Scale bars: 1 μm. ( H ) Lysosomal visualization using LAMP1 staining in differentiated WT and KO cells reveals an increased number and size (arrowheads) of lysosomes in the GLA -KO cells. Scale bar: 10 μm. ( I ) Quantification of lysosomal area ( n = 14), pH ( n = 12), and ROS production ( n = 8). ( J ) Seahorse XFp experiments confirm normal mitochondrial function in KO cells ( n = 8). OCR, oxygen consumption rate. ( K ) Mitochondrial import receptor subunit (TOM20) staining in WT and KO cells is equally abundant and normally distributed. TEM images confirm a normal mitochondrial ultrastructure in KO cells. Scale bars: 10 μm in immunofluorescence, 500 nm in electron microscopy. Violin plots indicate median (red) and upper and lower quartile (blue). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. One-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: The following antibodies were used in Western blot experiments: α-galactosidase (OriGene Technologies, TA336243), α-tubulin (Sigma-Aldrich, T9026), actinin 4 (Abcam, ab108198), p62 (Cell Signaling Technology, 5114), LC3 (Invitrogen, PA1-16931), SNCA (Santa Cruz Biotechnology, sc-12767), SCARB2 (Lifespan Biosciences, LS-B3225-0.05), TTYH3 (Origene, TA339554), MFEG8 (Sigma-Aldrich, HPA002807), CD63 (Proteintech, 25682-1-AP), DDP (Novus Biologicals, H00001678), PLSCR3 (Abcam, ab57554), GBA (Abcam, ab96246), and ITM2B (Abcam, ab119044).

Techniques: Transmission Assay, Electron Microscopy, Comparison, Control, Knock-Out, CRISPR, Western Blot, Expressing, Clone Assay, Activity Assay, Mass Spectrometry, Staining, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Septin4 promotes cell death in human colon cancer cells by interacting with BAX

doi: 10.7150/ijbs.44429

Figure Lengend Snippet: Septin4 participated in DOX-induced apoptosis of HCT116 cells. A. HCT116 cells were treated with DOX at 0, 0.005, 0.05, 0.2, 0.4, and 2 μmol/L for 48 h, to detect the expression of cleaved-caspase3 and cleaved-PARP1. B. Quantification of protein expression in A were shown as the means±S.D., *** P <0.001 C. The expression of PCNA and Septin4 was detected in the presence of DOX at 0, 0.005, 0.05, 0.2, 0.4, and 2 μmol/L for 48 h. D. Quantification of protein expression in C were shown as the means±S.D., ** P <0.01, *** P <0.001. E. The generation of ROS was detected by DHR staining under the treatment of 0, 0.005, 0.05, 0.2, 0.4, and 2 μmol/L DOX for 48 h. Scale bars, 100 μm. F. Quantitative analyses of the fluorescence intensity in E, data are shown as means±S.D., *** P <0.001. G. Statistical results of CCK8 analysis of HCT116 treated with DOX at 0, 0.005, 0.05, 0.2, 0.4, and 2 μmol/L for 48 h, data are shown as means±S.D., *** P <0.001.

Article Snippet: The membranes were incubated with blocking solution (TBST + 5% BSA) at room temperature for 1 h, then specific primary antibodies: goat polyclonal anti-Septin4 (ab166788, Abcam, UK), rabbit polyclonal anti-cleaved-caspase3 (19877-1-AP, Proteintech, USA), rabbit polyclonal anti-cleaved-PARP1 (5625S, Cell Signaling Technology, USA), rabbit polyclonal anti-BAX (50599-2-lg, Proteintech, USA), rabbit polyclonal anti-PCNA (10205-2-AP, Proteintech, USA), mouse monoclonal anti-Flag (GNI4110-FG, GNI, Japan), mouse monoclonal anti-GAPDH (10494-1-AP, Proteintech) or mouse monoclonal anti-β-actin (mAbcam 8226, Abcam, UK) were added and the blots were slowly shaken overnight at 4°C.

Techniques: Expressing, Staining, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: Septin4 promotes cell death in human colon cancer cells by interacting with BAX

doi: 10.7150/ijbs.44429

Figure Lengend Snippet: Overexpression of Septin4 enhanced sensitivity to DOX in colon cancer cells. A. HCT116 overexpressing Flag-Vector and Flag-Septin4 were treated with DOX at a concentration of 0.05 μmol/L for 48 h to detect the expression of cleaved-PARP1 and Flag-Septin4. B. Quantitative analysis of the expression of cleaved-PARP1 in A . Data were shown as the means±S.D., *** P <0.001. C. HCT116 overexpressing Flag-Vector and Flag-Septin4 were treated with DOX at a concentration of 0.05 μmol/L for 48 h to detect the expression of cleaved-caspase3 and Flag-Septin4. D. Quantitative analysis of the expression of cleaved-caspase3. Data were shown as the means±S.D., *** P <0.001. E. HCT116 overexpressing Flag-Vector and Flag-Septin4 were treated with DOX at a concentration of 0.05 μmol/L for 48 h to detect the expression of PCNA and Flag-Septin4. F. Quantitative analysis of the expression of PCNA in E . Data were shown as the means±S.D., *** P <0.001. G. The generation of ROS was detected by DHR staining in HCT116 overexpressing Flag-Vector and Flag-Septin4 treated with DOX at a concentration of 0.05 μmol/L for 48 h. Scale bars, 100 μm. H. Quantitative analyses of the fluorescence intensity in G . Data are shown as means±S.D., *** P <0.001. I. Statistical results of CCK8 assay of HCT116 overexpressing Flag-Vector and Flag-Septin4 under the DOX treatment at 0, 0.005, 0.05, 0.2, 0.4, and 2 μmol/L for 48 h. Data are shown as means±S.D., ** P <0.01, *** P <0.001.

Article Snippet: The membranes were incubated with blocking solution (TBST + 5% BSA) at room temperature for 1 h, then specific primary antibodies: goat polyclonal anti-Septin4 (ab166788, Abcam, UK), rabbit polyclonal anti-cleaved-caspase3 (19877-1-AP, Proteintech, USA), rabbit polyclonal anti-cleaved-PARP1 (5625S, Cell Signaling Technology, USA), rabbit polyclonal anti-BAX (50599-2-lg, Proteintech, USA), rabbit polyclonal anti-PCNA (10205-2-AP, Proteintech, USA), mouse monoclonal anti-Flag (GNI4110-FG, GNI, Japan), mouse monoclonal anti-GAPDH (10494-1-AP, Proteintech) or mouse monoclonal anti-β-actin (mAbcam 8226, Abcam, UK) were added and the blots were slowly shaken overnight at 4°C.

Techniques: Over Expression, Plasmid Preparation, Concentration Assay, Expressing, Staining, Fluorescence, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: Septin4 promotes cell death in human colon cancer cells by interacting with BAX

doi: 10.7150/ijbs.44429

Figure Lengend Snippet: Depletion of Septin4 resisted DOX-induced apoptosis in HCT116 cells. A. Expression of cleaved-PARP1 and cleaved-caspase3 in NC and Septin4-knockdown HCT116 cells were treated with DOX at 0.05 μmol/L for 48 h. B. Quantitative analysis of the expression of cleaved-PARP1 and cleaved-caspase3 in A . Data were shown as the means±S.D., *** P <0.001. C. ROS staining of the NC and shSeptin4 HCT116 cells were treated with DOX at 0.05 μmol/L for 48 h. Scale bars, 100 μm. D. Quantitative analyses of the fluorescence intensity in C . Data are shown as means±S.D., *** P <0.001. E. Statistical results of CCK8 analysis of NC and shSeptin4 HCT116 cells under 0.05 μmol/L DOX treatment for 48 h, *** P <0.001.

Article Snippet: The membranes were incubated with blocking solution (TBST + 5% BSA) at room temperature for 1 h, then specific primary antibodies: goat polyclonal anti-Septin4 (ab166788, Abcam, UK), rabbit polyclonal anti-cleaved-caspase3 (19877-1-AP, Proteintech, USA), rabbit polyclonal anti-cleaved-PARP1 (5625S, Cell Signaling Technology, USA), rabbit polyclonal anti-BAX (50599-2-lg, Proteintech, USA), rabbit polyclonal anti-PCNA (10205-2-AP, Proteintech, USA), mouse monoclonal anti-Flag (GNI4110-FG, GNI, Japan), mouse monoclonal anti-GAPDH (10494-1-AP, Proteintech) or mouse monoclonal anti-β-actin (mAbcam 8226, Abcam, UK) were added and the blots were slowly shaken overnight at 4°C.

Techniques: Expressing, Knockdown, Staining, Fluorescence