Journal: Biomedicines

Article Title: miR-210 Regulates Apoptotic Cell Death during Cellular Hypoxia and Reoxygenation in a Diametrically Opposite Manner

doi: 10.3390/biomedicines10010042

Figure Lengend Snippet: List of antibodies used in the study.

Article Snippet: APAF1 , IP , 5 μg , Rabbit , Novus Biologicals , NBP1-77000 , AB_11008194.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: CASP9 (caspase 9) is essential for autophagosome maturation through regulation of mitochondrial homeostasis.

doi: 10.1080/15548627.2019.1695398

Figure Lengend Snippet: Figure 5. APAF1 is not required for CASP9 cleavage and autophagy induction following INS withdrawal in HCN cells. (A) Western blotting analysis for validation of Apaf1 KO. (B) Western blotting analysis for evaluation of the effect of Apaf1 KO on CASP9 cleavage after treatment with STS (0.5 μM, 6 h). The blots shown are representative of 3 experiments with similar results. (C) CASP9 activity after INS withdrawal for 3 h in sgCon and sgApaf1 cells (n = 4). (D) Western blotting analysis of MAP1LC3B after INS withdrawal for 6 h in sgCon and sgApaf1 cells. BafA1 (10 nM) was added 1 h before cell harvest. Graph, quantification of MAP1LC3B-II normalized to ACTB (n = 3). (E) Cell death rate in sgCon, sgApaf1 and sgCasp9 cells following INS withdrawal for 24 h (n = 4). (F) Cell death rate in sgCon, sgApaf1 and sgCasp9 cells after treatment of STS for 12 h (n = 4). **P < 0.01, ***P < 0.001; ns, not significant.

Article Snippet: Antibodies against the following proteins were used: MAP1LC3B (NB100-2220), ATG5 (NB110-53818), and APAF1 (NBP1-76999) from Novus Biologicals; GABARAPL1 (11010-1-AP) and GABARAPL2 (18724-1-AP) from Proteintech; FLAG (F3165) from SigmaAldrich; GABARAP (13733S), p-MTOR S2448 (5536), MTOR (2972S), p-ULK1 S757 (14202), ULK1 (8054S), BECN1 (3738S), ATG3 (3415), ATG7 (8558), ATG16L1 (8089), pro-CASP3 (9662), cleaved CASP3 (9661), CASP9 (9508), cleaved CASP9 (9595 for human, 9507 for rat), PARP1 (9542), TUBB (2146), VDAC (4866), p-DNM1L S616 (4494), and MFN2 (9482) from Cell Signaling Technology; p-BECN1 S15 (254515) from Abbiotec; p-DNM1L S637 (orb 127984) from Biorbyt; RAB7 (sc376362), DNM1L (sc-32898), MFN1 (sc-166644) and horseradish peroxidase-conjugated ACTB/β-actin (sc-47778) from Santa Cruz Biotechnology.

Techniques: Western Blot, Biomarker Discovery, Activity Assay

Journal: Biomedicines

Article Title: miR-210 Regulates Apoptotic Cell Death during Cellular Hypoxia and Reoxygenation in a Diametrically Opposite Manner

doi: 10.3390/biomedicines10010042

Figure Lengend Snippet: List of antibodies used in the study.

Article Snippet: The APAF1 antibody blocking peptide (Novus Biologicals/Bio-Techne, Abingdon, United Kingdom, Catalogue # NBP1-77000PEP), cytochrome c antibody blocking peptide (Cell Signaling Technology, Danvers, MA, USA, Catalogue # 1033), and the procaspase-9 antibody blocking peptide (Novus Biologicals/Bio-Techne, Abingdon, United Kingdom, Catalogue # NBP1-76961PEP), corresponding to the specific epitopes recognized by the respective detection antibodies, were used for the peptide blocking assays .

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: miR-210 Regulates Apoptotic Cell Death during Cellular Hypoxia and Reoxygenation in a Diametrically Opposite Manner

doi: 10.3390/biomedicines10010042

Figure Lengend Snippet: miR-210 reduced the hypoxia (18 h)-induced apoptosome formation. ( A – F ) Co-immunoprecipitation (Co-IP) coupled with tandem ELISA determining the quantitative abundance of APAF1 and cytochrome c in the procaspase-9 immunoprecipitates were performed as a surrogate measure of the abundance of the apoptosome formation. ( A – D ) ELISA immunoassays demonstrated that overexpression of endogenous miR-210 significantly attenuated the hypoxia-induced APAF1 ( A inset c) and cytochrome c ( B inset c) abundance in the procaspase-9 immunoprecipitates, whereas the ectopic expression of the miR-210-3p decoy/inhibitor significantly enhanced the hypoxia-induced APAF1 ( C inset c) and cytochrome c ( D inset c) abundance in procaspase-9 immunoprecipitates. ( E , F ) ELISA determining the quantitative abundance of procaspase-9 ( E inset c, F inset c) unequivocally showed equitable abundance of procaspase-9 in the procaspase-9 immunoprecipitates. (( A ) inset b—( F ) inset b) ELISA determining the quantitative abundance of APAF1 (inset b in ( A , C )), cytochrome c (inset b in ( B , D )), and procaspase-9 (inset b in ( E , F )) in the rabbit IgG immunoprecipitates demonstrated the specificity of the procaspase-9 Co-IP assays. (( A ) inset a—( F ) inset a) ELISA determining the quantitative abundance of APAF1 (inset a in ( A , C )), cytochrome c (inset a in ( B , D )), and procaspase-9 (inset a in ( E , F )) in the native lysates serving as input (5%). miR-210 expression levels in the respective cell lysate- inputs were determined by the miR-210 hybridization immunoassay (as described in the ) and are reported in . Data are expressed as experimental blank-corrected absorbances (O.D) measured at λ 570 (570 nm). Data are expressed as mean ± S.D from three technical replicates for each of the four biological replicates belonging to each experimental group ( n = 4); * p ≤ 0.05; ** p ≤ 0.01; ns: not significant ( p > 0.05); OE: miR-210 overexpression; KD: miR-210-3p decoy/inhibitor; H-R: hypoxia-reoxygenation; O.D: optical density; S.D: standard deviation.

Article Snippet: The APAF1 antibody blocking peptide (Novus Biologicals/Bio-Techne, Abingdon, United Kingdom, Catalogue # NBP1-77000PEP), cytochrome c antibody blocking peptide (Cell Signaling Technology, Danvers, MA, USA, Catalogue # 1033), and the procaspase-9 antibody blocking peptide (Novus Biologicals/Bio-Techne, Abingdon, United Kingdom, Catalogue # NBP1-76961PEP), corresponding to the specific epitopes recognized by the respective detection antibodies, were used for the peptide blocking assays .

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay, Over Expression, Expressing, Hybridization, Standard Deviation

Journal: Biomedicines

Article Title: miR-210 Regulates Apoptotic Cell Death during Cellular Hypoxia and Reoxygenation in a Diametrically Opposite Manner

doi: 10.3390/biomedicines10010042

Figure Lengend Snippet: miR-210 exacerbated the hypoxia-reoxygenation (H-R)-induced apoptosome formation. ( A – D ) ELISA immunoassays demonstrated that overexpression of miR-210 significantly augmented the H-R-induced APAF1 (( A ) inset c) and cytochrome c (( B ) inset c) abundance in the procaspase-9 immunoprecipitates, whereas the ectopic expression of the miR-210-3p decoy/inhibitor significantly attenuated the H-R-induced APAF1 (( C ) inset c) and cytochrome c (( D ) inset c) abundance in procaspase-9 immunoprecipitates. ( E , F ) ELISA determining the quantitative abundance of procaspase-9 (( E ) inset c, ( F ) inset c) unequivocally showed equitable abundance of procaspase-9 in the procaspase-9 immunoprecipitates. (( A ) inset b—( F ) inset b) ELISA determining the quantitative abundance of APAF1 (inset b in ( A , C )), cytochrome c (inset b in ( B , D )), and procaspase-9 (inset b in ( E , F )) in the rabbit IgG immunoprecipitates demonstrated the specificity of the procaspase-9 Co-IP assays. (( A ) inset a—( F ) inset a) ELISA determining the quantitative abundance of APAF1 (inset a in ( A , C )), cytochrome c (inset a in ( B , D )), and procaspase-9 (inset a in ( E , F )) in the native lysates serving as input (5%). miR-210 expression levels in the respective cell lysate-inputs were determined by the miR-210 hybridization immunoassay (as described in the ) and are reported in . Data are expressed as experimental blank-corrected absorbances (O.D) measured at λ 570 (570 nm). Data are expressed as mean ± S.D from three technical replicates for each of the four biological replicates belonging to each experimental group ( n = 4).** p ≤ 0.01; **** p ≤ 0.0001; ns: not significant ( p > 0.05); OE: miR-210 overexpression; KD: miR-210-3p decoy/inhibitor; H-R: hypoxia-reoxygenation; O.D: optical density; S.D: standard deviation.

Article Snippet: The APAF1 antibody blocking peptide (Novus Biologicals/Bio-Techne, Abingdon, United Kingdom, Catalogue # NBP1-77000PEP), cytochrome c antibody blocking peptide (Cell Signaling Technology, Danvers, MA, USA, Catalogue # 1033), and the procaspase-9 antibody blocking peptide (Novus Biologicals/Bio-Techne, Abingdon, United Kingdom, Catalogue # NBP1-76961PEP), corresponding to the specific epitopes recognized by the respective detection antibodies, were used for the peptide blocking assays .

Techniques: Enzyme-linked Immunosorbent Assay, Over Expression, Expressing, Co-Immunoprecipitation Assay, Hybridization, Standard Deviation