cleaved casp3 mouse mab  (Bioss)

Journal: Oncology Letters

Article Title: Transferrin receptor serves a role in promoting PANoptosis in thyroid cancer

doi: 10.3892/ol.2025.15394

Figure Lengend Snippet: TFRC contributes to PANoptosis in TC cells. Effects of (A) PGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro and (B) shRNA transfection on the expression of TFRC mRNA in BCPAP and K1 cells, assessed using reverse transcription-quantitative PCR assay. (C) PI/Calcein-AM staining was performed to assess the effect of TFRC on cell death in BCPAP (OV-NC and OV-TFRC groups) and K1 (KD-NC and KD-TFRC groups) cells. PI (red) and Calcein-AM (green) label dead and viable cells, respectively. (D) YO-PRO-1/PI staining was performed to evaluate pyroptosis, apoptosis and necroptosis in BCPAP and K1 cells following exogenous modulation of TFRC expression. YO-PRO-1 (green) indicates apoptotic or necroptotic cells, whereas PI (red) labels necroptotic or pyroptotic cells. (E) Western blot analysis of markers associated with pyroptosis (cleaved-CASP1/CASP1), apoptosis (cleaved-CASP3/CASP3) and necroptosis (pMLKL/tMLKL and tRIPK3), with corresponding quantitative analyses. TFRC, transferrin receptor; sh, short hairpin; OV, overexpression; NC, negative control; KD, knockdown; CASP; caspase; MLKL, mixed lineage kinase domain like pseudokinase; pMLKL, phospho-MLKL; tMLKL, total-MLKL; tRIPK3, total receptor interacting serine/threonine kinase 3.

Article Snippet: Membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with the following primary antibodies overnight at 4°C: Vinculin mouse mAb (1:50,000; cat. no. 66305-1-Ig; Proteintech Group, Inc.), CASP3 mouse mAb (1:2,000; cat. no. bsm-33284M; BIOSS), cleaved-CASP3 mouse mAb (1:2,000; cat. no. bsm-33199M; BIOSS), CASP1 rabbit pAb (1:2,000; cat. no. 22915-1-AP; Proteintech Group, Inc.), cleaved-CASP1 rabbit pAb (1:3,000; cat. no. PA5-77886; Thermo Fisher Scientific, Inc.), phospho-(p)MLKL rabbit pAb (1:2,000; cat. no. PA5-105677; Thermo Fisher Scientific, Inc.), total-(t)MLKL mouse mAb (1:50,000; cat. no. 66675-1-Ig; Proteintech Group, Inc.) and tRIPK3 rabbit pAb (1:10,000; cat. no. 29080-1-AP; Proteintech Group, Inc.).

Techniques: shRNA, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Western Blot, Over Expression, Negative Control, Knockdown

Journal: Oncology Letters

Article Title: Transferrin receptor serves a role in promoting PANoptosis in thyroid cancer

doi: 10.3892/ol.2025.15394

Figure Lengend Snippet: TFRC contributes to PANoptosis in TC cells. Effects of (A) PGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro and (B) shRNA transfection on the expression of TFRC mRNA in BCPAP and K1 cells, assessed using reverse transcription-quantitative PCR assay. (C) PI/Calcein-AM staining was performed to assess the effect of TFRC on cell death in BCPAP (OV-NC and OV-TFRC groups) and K1 (KD-NC and KD-TFRC groups) cells. PI (red) and Calcein-AM (green) label dead and viable cells, respectively. (D) YO-PRO-1/PI staining was performed to evaluate pyroptosis, apoptosis and necroptosis in BCPAP and K1 cells following exogenous modulation of TFRC expression. YO-PRO-1 (green) indicates apoptotic or necroptotic cells, whereas PI (red) labels necroptotic or pyroptotic cells. (E) Western blot analysis of markers associated with pyroptosis (cleaved-CASP1/CASP1), apoptosis (cleaved-CASP3/CASP3) and necroptosis (pMLKL/tMLKL and tRIPK3), with corresponding quantitative analyses. TFRC, transferrin receptor; sh, short hairpin; OV, overexpression; NC, negative control; KD, knockdown; CASP; caspase; MLKL, mixed lineage kinase domain like pseudokinase; pMLKL, phospho-MLKL; tMLKL, total-MLKL; tRIPK3, total receptor interacting serine/threonine kinase 3.

Article Snippet: Membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with the following primary antibodies overnight at 4°C: Vinculin mouse mAb (1:50,000; cat. no. 66305-1-Ig; Proteintech Group, Inc.), CASP3 mouse mAb (1:2,000; cat. no. bsm-33284M; BIOSS), cleaved-CASP3 mouse mAb (1:2,000; cat. no. bsm-33199M; BIOSS), CASP1 rabbit pAb (1:2,000; cat. no. 22915-1-AP; Proteintech Group, Inc.), cleaved-CASP1 rabbit pAb (1:3,000; cat. no. PA5-77886; Thermo Fisher Scientific, Inc.), phospho-(p)MLKL rabbit pAb (1:2,000; cat. no. PA5-105677; Thermo Fisher Scientific, Inc.), total-(t)MLKL mouse mAb (1:50,000; cat. no. 66675-1-Ig; Proteintech Group, Inc.) and tRIPK3 rabbit pAb (1:10,000; cat. no. 29080-1-AP; Proteintech Group, Inc.).

Techniques: shRNA, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Western Blot, Over Expression, Negative Control, Knockdown

Journal: Free radical biology & medicine

Article Title: Maternal riboflavin deficiency causes embryonic defects by activating ER stress-induced hepatocyte apoptosis pathway.

doi: 10.1016/j.freeradbiomed.2024.09.002

Figure Lengend Snippet: Fig. 3. Real-time PCR and Western blot analyses verify gene and protein expression of duck embryos. (A) Results of RD on the relative gene expression of CHOP, GRP78, ATF6, ERO1, BAX, BCL2, BAX/BCL2, CASP3, SOD1, and SOD2. Each treatment is performed in six replicates (n = 6). (B–C) Western blots revealed that RD on the protein expression of CHOP, GRP78, CASP3, BCL2, SOD1, and SOD2. Each treatment is performed in five replicates (n = 5)Values are expressed as means ± SD. Different letters indicate significant differences (P < 0.05). Abbreviations: RD, riboflavin deficiency; CON, control (riboflavin sufficiency); CHOP, C/EBP homologous protein; GRP78, 78 kDa glucose-regulated protein; ATF6, activating transcription factor 6; ERO1, endoplasmic reticulum oxidoreductin-1; BAX, BCL2-associated X, apoptosis regulator; BCL2, B-cell lymphoma 2, apoptosis regulator, CASP3, Caspase 3; SOD1, Superoxide dismutase (Cu-Zn); SOD2, Superoxide dismutase (Mn).

Article Snippet: The full list of primary and secondary antibodies is given below: CHOP (#2895, 1:1000, CST, Danvers, MA), 78 kDa glucose-regulated protein (GRP78, #3177T, 1:1000, CST), activating transcription factor 6 (ATF6, #D4Z8V, 1:1000, CST), CASP3 (#9668T, 1:1000, CST), B-cell lymphoma 2, apoptosis regulator (BCL2, #A11313, 1:1000, ABclonal, Wuhan, China), Superoxide dismutase (Cu-Zn) (SOD1, #A0274, 1:1000, ABclonal), Superoxide dismutase (Mn) (SOD2, #A1340, 1:1000, ABclonal), β-tubulin (#HX1829, 1:5000, Huaxingbio, Beijing, China), Goat Anti-Rabbit IgG (#SE131, 1:5000, Solarbio), and goat Anti-Rat IgG (#SE134, 1:5000, Solarbio).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Gene Expression, Control

Journal: Free radical biology & medicine

Article Title: Maternal riboflavin deficiency causes embryonic defects by activating ER stress-induced hepatocyte apoptosis pathway.

doi: 10.1016/j.freeradbiomed.2024.09.002

Figure Lengend Snippet: Fig. 5. Real-time PCR and Western blot analyses verify gene and protein expression of PDEH. (A) Results of RD on the relative gene expression of CHOP, GRP78, ATF6, ERO1, BAX, BCL2, BAX/BCL2, CASP3, SOD1, and SOD2. Each treatment is performed in six replicates (n = 6). (B–C) Western blots revealed RD on the protein expression of CHOP, GRP78, CASP3, BCL2, SOD1, and SOD2. Each treatment is performed in five replicates (n = 5).Values are expressed as means ± SD. Different letters indicate significant differences (P < 0.05). Abbreviations: RD, riboflavin deficiency; CON, control (riboflavin sufficiency); PDEH, primary duck embryonic hepatocytes; CHOP, C/EBP homologous protein; GRP78, 78 kDa glucose-regulated protein; ATF6, activating transcription factor 6; ERO1, endoplasmic reticulum oxidoreductin-1; BAX, BCL2-associated X, apoptosis regulator; BCL2, B-cell lymphoma 2, apoptosis regulator, CASP3, Caspase 3; SOD1, Superoxide dismutase (Cu-Zn); SOD2, Superoxide dismutase (Mn).

Article Snippet: The full list of primary and secondary antibodies is given below: CHOP (#2895, 1:1000, CST, Danvers, MA), 78 kDa glucose-regulated protein (GRP78, #3177T, 1:1000, CST), activating transcription factor 6 (ATF6, #D4Z8V, 1:1000, CST), CASP3 (#9668T, 1:1000, CST), B-cell lymphoma 2, apoptosis regulator (BCL2, #A11313, 1:1000, ABclonal, Wuhan, China), Superoxide dismutase (Cu-Zn) (SOD1, #A0274, 1:1000, ABclonal), Superoxide dismutase (Mn) (SOD2, #A1340, 1:1000, ABclonal), β-tubulin (#HX1829, 1:5000, Huaxingbio, Beijing, China), Goat Anti-Rabbit IgG (#SE131, 1:5000, Solarbio), and goat Anti-Rat IgG (#SE134, 1:5000, Solarbio).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Gene Expression, Control

Journal: Free radical biology & medicine

Article Title: Maternal riboflavin deficiency causes embryonic defects by activating ER stress-induced hepatocyte apoptosis pathway.

doi: 10.1016/j.freeradbiomed.2024.09.002

Figure Lengend Snippet: Fig. 7. 4-PBA reduced RD-induced high expression of ER stress and apoptosis markers (A) Results of mRNA expression of PDEH treated with 4-PBA. Representative relative gene expression of CHOP, GRP78, ATF6, ERO1, BAX, BCL2, BAX/BCL2, and CASP3. Each treatment is performed in six replicates (n = 6). (B–C) Results of protein expression of PDEH treated with 4-PBA. Western blot analysis of CHOP, GRP78, BCL2, and CASP3 protein expression. Each treatment is performed from four to five replicates (n = 4～5). The blue, yellow, and red bars represent the RD-BC, RD treated with 5 mM 4-PBA, and CON-BC groups, respectively. Values are expressed as means ± SD. Different letters indicate significant differences (P < 0.05). Abbreviations: RD, riboflavin deficiency; CON, control (riboflavin sufficiency); BC, blank control; PDEH, primary duck embryonic hepatocytes; 4-PBA, 4-phenyl butyric acid; CHOP, C/EBP homologous protein; GRP78, 78 kDa glucose-regulated protein; ATF6, activating transcription factor 6; ERO1, endoplasmic reticulum oxidoreductin-1; BAX, BCL2-associated X, apoptosis regulator; BCL2, B-cell lymphoma 2, apoptosis regulator, CASP3, Caspase 3.

Article Snippet: The full list of primary and secondary antibodies is given below: CHOP (#2895, 1:1000, CST, Danvers, MA), 78 kDa glucose-regulated protein (GRP78, #3177T, 1:1000, CST), activating transcription factor 6 (ATF6, #D4Z8V, 1:1000, CST), CASP3 (#9668T, 1:1000, CST), B-cell lymphoma 2, apoptosis regulator (BCL2, #A11313, 1:1000, ABclonal, Wuhan, China), Superoxide dismutase (Cu-Zn) (SOD1, #A0274, 1:1000, ABclonal), Superoxide dismutase (Mn) (SOD2, #A1340, 1:1000, ABclonal), β-tubulin (#HX1829, 1:5000, Huaxingbio, Beijing, China), Goat Anti-Rabbit IgG (#SE131, 1:5000, Solarbio), and goat Anti-Rat IgG (#SE134, 1:5000, Solarbio).

Techniques: Expressing, Gene Expression, Western Blot, Control

Journal: Free radical biology & medicine

Article Title: Maternal riboflavin deficiency causes embryonic defects by activating ER stress-induced hepatocyte apoptosis pathway.

doi: 10.1016/j.freeradbiomed.2024.09.002

Figure Lengend Snippet: Fig. 9. CHOP knockdown transfection reduced RD-induced high expression of apoptosis markers (A) Results of mRNA expression of PDEH treated with CHOP siRNA transfection. Representative relative gene expression of CHOP, CASP3, ERO1, BAX, BCL2, and BAX/BCL2. Each treatment is performed in six replicates (n = 6). (B–C) Results of protein expression of PDEH treated with CHOP siRNA transfection. Western blot analysis of CHOP, BCL2, and CASP3 proteins expression. Each treatment is performed from four to ten replicates (n = 4～10). The blue, yellow, red, and green bars represent the RD-BC, RD-NC, RD-siCHOP, and CON-BC groups, respectively. Values are expressed as means ± SD. Different letters indicate significant differences (P < 0.05). Abbreviations: RD, riboflavin deficiency; CON, control (riboflavin sufficiency); BC, blank control; NC, negative control siRNA transfection; siCHOP, CHOP siRNA transcription; PDEH, primary duck embryonic hepatocytes; CHOP, C/EBP homologous protein; CASP3, Caspase 3; ERO1, endoplasmic reticulum oxidoreductin-1; BAX, BCL2-associated X, apoptosis regulator; BCL2, B-cell lymphoma 2, apoptosis regulator.

Article Snippet: The full list of primary and secondary antibodies is given below: CHOP (#2895, 1:1000, CST, Danvers, MA), 78 kDa glucose-regulated protein (GRP78, #3177T, 1:1000, CST), activating transcription factor 6 (ATF6, #D4Z8V, 1:1000, CST), CASP3 (#9668T, 1:1000, CST), B-cell lymphoma 2, apoptosis regulator (BCL2, #A11313, 1:1000, ABclonal, Wuhan, China), Superoxide dismutase (Cu-Zn) (SOD1, #A0274, 1:1000, ABclonal), Superoxide dismutase (Mn) (SOD2, #A1340, 1:1000, ABclonal), β-tubulin (#HX1829, 1:5000, Huaxingbio, Beijing, China), Goat Anti-Rabbit IgG (#SE131, 1:5000, Solarbio), and goat Anti-Rat IgG (#SE134, 1:5000, Solarbio).

Techniques: Knockdown, Transfection, Expressing, Gene Expression, Western Blot, Control, Negative Control

Journal: Free radical biology & medicine

Article Title: Maternal riboflavin deficiency causes embryonic defects by activating ER stress-induced hepatocyte apoptosis pathway.

doi: 10.1016/j.freeradbiomed.2024.09.002

Figure Lengend Snippet: Fig. 12. 4-PBA reduced RD-induced high expression of ER stress and apoptosis markers in duck embryos. (A) Results of mRNA expression of duck embryos treated with 4-PBA. Representative relative gene expression of CHOP, GRP78, ATF6, ERO1, BAX, BCL2, BAX/BCL2, and CASP3. Each treatment is performed from 11 to 12 replicates (n = 11~12). (B–C) Results of protein expression of duck embryos treated with 4-PBA. Western blot analysis of CHOP, GRP78, BCL2, and CASP3 protein expression. Each treatment is performed from four to five replicates (n = 4～5).The blue, yellow, and red bars represent the RD-BC, injected with 200 mg/(kg egg weight) 4-PBA in the fertilized eggs of the RD (RD 4-PBA), and CON-BC groups, respectively.Values are expressed as means ± SD. Different letters indicate significant differences (P < 0.05).Abbreviations: RD, riboflavin deficiency; CON, control (riboflavin sufficiency); BC, blank control; PDEH, primary duck embryonic hepatocytes; 4-PBA, 4-phenyl butyric acid; CHOP, C/EBP homologous protein; GRP78, 78 kDa glucose-regulated protein; ATF6, activating transcription factor 6; ERO1, endoplasmic reticulum oxidoreductin-1; BAX, BCL2-associated X, apoptosis regulator; BCL2, B-cell lymphoma 2, apoptosis regulator, CASP3, Caspase 3.

Article Snippet: The full list of primary and secondary antibodies is given below: CHOP (#2895, 1:1000, CST, Danvers, MA), 78 kDa glucose-regulated protein (GRP78, #3177T, 1:1000, CST), activating transcription factor 6 (ATF6, #D4Z8V, 1:1000, CST), CASP3 (#9668T, 1:1000, CST), B-cell lymphoma 2, apoptosis regulator (BCL2, #A11313, 1:1000, ABclonal, Wuhan, China), Superoxide dismutase (Cu-Zn) (SOD1, #A0274, 1:1000, ABclonal), Superoxide dismutase (Mn) (SOD2, #A1340, 1:1000, ABclonal), β-tubulin (#HX1829, 1:5000, Huaxingbio, Beijing, China), Goat Anti-Rabbit IgG (#SE131, 1:5000, Solarbio), and goat Anti-Rat IgG (#SE134, 1:5000, Solarbio).

Techniques: Expressing, Gene Expression, Western Blot, Injection, Control

Journal: Cell reports

Article Title: Hierarchical cell-type-specific functions of caspase-11 in LPS shock and antibacterial host defense

doi: 10.1016/j.celrep.2021.109012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal Anti-mouse Casp3 (cleaved) (Clone 5A1E) , Cell Signaling Technology , Cat# 9664; RRID:AB_2070042.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Virus, Recombinant, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Mutagenesis, Software

Journal: Nature biomedical engineering

Article Title: AND-gate contrast agents for enhanced fluorescence-guided surgery

doi: 10.1038/s41551-020-00616-6

Figure Lengend Snippet: a, Structures of the dual orthogonal protease substrate AND-Gate probes and corresponding negative controls. DEATH-CAT 1 contains the natural amino acid (L)-Glu while the DEATH-CAT 2 contains a non-natural (D)-Glu linker (blue). The negative control probes contain either a (D)-Asp in the P1 position of the Casp3 sequence ( (D)-Asp 1 ) or (D)-Phe in the P2 position of the Cat sequence ( (D)-Phe 1 ) to block proteolytic cleavage by the respective protease. b , Progress curve of DEATH-CAT 2 incubated with CatL followed by Casp3 compared to negative control probes (D)-Phe 2 and (D)-Asp 2 and with Casp3 only (10 nM). All substrates were used at 10 μM. c , Progress curves as in ( b ) except Casp3 was added first followed by CatL. The single substrate probe 6QC was incubated with CatL alone. d , Progress curve over 2 hours incubation of DEATH-CAT 2 compared to 6QC and corresponding negative controls, (D)-Phe-2 and (D)-Asp-2 , in either buffer or tumor lysate generated from 4T1 breast tumors in BALB/c mice. e , Representative fluorescent microscopy images of 4T1 cells co-cultured with RAW macrophages (1:1 ratio) labeled with either the single-parameter cathepsin probe 6QC or the DEATH-CAT 2 probe. Cells were incubated with either DMSO or etoposide (5 μM) for 24 h prior to addition of probes (1 μM). After 2 h, Hoechst stain was added to visualize nuclei and the cells were imaged. The Cy5 signal is shown in gray scale in the left panels. For merged images on the right, brightfield is in grayscale, blue is nuclear staining, and red is the Cy5 probe signal. Representative images of the negative controls (D)Phe 2 , (D)Asp 2 , and Casp3-QC can be found in the . Scale bars are 20 μm. f , Scatter plot of fluorescent signal from ( e ) shown as the fold change of corrected total cellular fluorescence normalized to cells incubated with DMSO and the DEATH-CAT 2 probe (Mann-Whitney Test, two-tailed: *** p < 0.0001). Sample mean ± S.D., sample size (n) are as follows– 6QC (DMSO): 13.6 ± 12.1, 416; 6QC (Etoposide): 23.9 ± 19.6, 266; DEATH-CAT 2 (DMSO): 1.0 ± 0.6, 398; DEATH-CAT 2 (Etoposide): 2.7 ± 1.8, 462; (D)-Asp 2 (DMSO): 0.7 ± 0.5, 599; (D)-Asp 2 (Etoposide): 0.8 ± 0.6, 246; (D)-Phe 2 (DMSO): 0.2 ± 0.1, 402; (D)-Phe 2 (Etoposide): 0.5 ± 0.4, 269; Casp3-QC (DMSO): 0.2 ± 0.1, 226; Casp3-QC (Etoposide): 0.5 ± 0.4, 205. The data presented were acquired from three biological replicates. Total images taken for each condition includes three field of views per well from six separate wells.

Article Snippet: Samples were washed 3X with 0.5% w/v BSA, then tissues were outlined with a hydrophobic pen, followed by incubation with 1:2,500 dilution of primary antibodies for CD68 (rat anti-mouse, Bio-Rad), cleaved Casp3 (rabbit anti-mouse, Cell Signaling), or FAP (rabbit anti-mouse, R&D Systems) in 0.5% w/v BSA in 1X PBS for 24 h at 4 °C.

Techniques: Negative Control, Sequencing, Blocking Assay, Incubation, Generated, Microscopy, Cell Culture, Labeling, Staining, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Nature biomedical engineering

Article Title: AND-gate contrast agents for enhanced fluorescence-guided surgery

doi: 10.1038/s41551-020-00616-6

Figure Lengend Snippet: a , Scatter plot of average fluorescent signal within splayed tumors from all mice 2 h post injection of each probe (20 nmol, I.V.). (One-way ANOVA, Tukey’s Multiple Comparison Test, multiplicity adjusted p-values: **p = 0.002, ***p < 0.0001). Sample mean ± S.D., sample size (n) are as follows– DEATH-CAT 2 : 2.3 ± 0.7, 12; 6QC : 1.6 ± 0.2, 12; Casp3-QC : 1.0 ± 0.2, 10; (D)-Phe 2 : 0.7 ± 0.1, 10; (D)-Asp 2 : 0.7 ± 0.2, 12. b , Immunofluorescence microscopy images of 4T1 tumor sections from mice treated with DEATH-CAT 2 and stained for cleaved Casp3 and CD68 using specific antibodies. Single channel images are in grayscale. Merge images are as follows: Cy5 (probe) is magenta, CD68 is yellow, cleaved Casp3 is green, DAPI is blue. The same contrast and brightness settings were used to process each image. Top column images scale bar is 50 μm and lower column scale bar is 10 μm. Images of additional examples of stained 4T1 tumor sections can be found in the and are representative of fifty field of views. c , Images of excised organs of mice (2 h post injection). Fluorescent signal is displayed as rainbow plots and is normalized between images. Images are representative of each cohort. d , Scatter plot of the fold change in fluorescence signal compared to tumor signals for each tissue type in mice treated with the indicated probes. (Student’s T-test, two-tailed, Benforroni-Holm Procedure, unadjusted p-values: **p = 0.01, ***p < 0.001). Sample mean ± S.D., sample size (n) are as follows– 6QC (liver): 0.9 ± 0.2, 6; DEATH-CAT 2 (liver): 0.4 ± 0.2, 6; 6QC (kidneys): 1.7 ± 0.4, 12; DEATH-CAT 2 (kidneys): 0.6 ± 0.3, 12; 6QC (lungs): 0.3 ± 0.1, 12; DEATH-CAT 2 (lungs): 0.2 ± 0.1, 12; 6QC (fat): 0.4 ± 0.2, 12; DEATH-CAT 2 (fat): 0.3 ± 0.1, 12. Overlay images of fluorescent signal and brightfield of all live mice, splayed tumors, and excised organs from each cohort can be found in the . The data presented were acquired from at least 10 biological replicates.

Article Snippet: Samples were washed 3X with 0.5% w/v BSA, then tissues were outlined with a hydrophobic pen, followed by incubation with 1:2,500 dilution of primary antibodies for CD68 (rat anti-mouse, Bio-Rad), cleaved Casp3 (rabbit anti-mouse, Cell Signaling), or FAP (rabbit anti-mouse, R&D Systems) in 0.5% w/v BSA in 1X PBS for 24 h at 4 °C.

Techniques: Injection, Comparison, Immunofluorescence, Microscopy, Staining, Fluorescence, Two Tailed Test

Journal: European Journal of Human Genetics

Article Title: Key apoptotic genes APAF1 and CASP9 implicated in recurrent folate-resistant neural tube defects

doi: 10.1038/s41431-017-0025-y

Figure Lengend Snippet: Functional studies examining expression of CASP9 and downstream apoptotic pathway activation. a Quantification of western blot of CASP9 in control and affected fetuses II-1 and II-3 in Family 1 and affected fetus II-10 in Family 2. Level of CASP9 protein is significantly reduced in all fetal fibroblast cells and almost absent in Family 2 fetus II-10 with the CASP9 variants in trans (*p < 0.05, **p < 0.01, ***p < 0.001). b Quantification of activation of downstream apoptotic factors. Level of cleaved CASP9, CASP3, and PARP was significantly reduced in fibroblast cells from all three fetuses as compared with control (*p < 0.01, **p < 0.001, ***p < 0.0001). c TUNEL assay in control and fetal fibroblasts. Significant reduction in apoptosis was observed in NTD fetus cell lines compared to control (*p < 0.01, **p < 0.001). Student’s t-test was used to compare data from patient and control fibroblasts. Graphs represent the averages and standard deviation for experiments performed in triplicate

Article Snippet: Primary antibodies used to detect target proteins in 20 μg cell lysate were the following: anti-CASP9 antibody diluted 1:1000 (Cell Signaling Technology, Danvers, MA, USA, #9502), anti-PARP antibody diluted to 1:1000 (Cell Signaling Technology, #9542), and anti-CASP3 antibody diluted to 1:1000 (Cell Signaling Technology, #9668), and anti-GADPH antibody diluted to 1:10,000 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, #sc-32233).

Techniques: Functional Assay, Expressing, Activation Assay, Western Blot, Control, TUNEL Assay, Standard Deviation