Journal: Frontiers in Cell and Developmental Biology

Article Title: Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle

doi: 10.3389/fcell.2021.710864

Figure Lengend Snippet: Expression of alpha/beta hydrolase domain-containing protein 2 (ABHD2) in the mouse ovary. (a) Immunohistochemical (IHC) staining of a 1-month-old superovulated wild-type mouse ovary shows ABHD2 in the stromal cells (1) and in cells of the corpus luteum (CL) (2), while the protein is not present in the granulosa cells and oocytes of developing follicles, demarcated by staining of the surrounding smooth muscle layer (b) , and (3) Stromal cells of 1-week-old pre-pubertal mouse ovaries also display ABHD2 staining while (c) shows similar expression levels of Abhd2 before and after the first round of ovulation, as detected in 21- and 28-day-old mice ovaries. The expression levels increase after ovulation, as seen in ovaries at estrus and after superovulation. Samples ( n = 3 for each timepoint) were collected from 3-month-old females in proestrus and estrus and 1-month-old superovulated females. Expression levels measured by qPCR were normalized to the expression of ribosomal protein L19 ( Rpl19 ) and peptidylprolyl isomerase A ( Ppia ). Statistical significance was calculated using the unpaired t -test, and the significance of changes is indicated as follows: * p ≤ 0.05.

Article Snippet: The samples were analyzed by Western blotting, using a rabbit polyclonal anti-ABHD2 antibody (1:10,000 dilution, Proteintech Group, 14039-1-AP) and a peroxidase-conjugated anti-rabbit secondary antibody (1:15,000 dilution, Abcam, ab6721).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle

doi: 10.3389/fcell.2021.710864

Figure Lengend Snippet: Generation of the Abhd2 KO mouse line. (a) Schematic picture of the Mus musculus Abhd2 gene with untranslated regions marked in gray. Deletion of exon 6 (in red) was performed by CRISPR/Cas9, targeting sequences in the surrounding introns. The sequences of the two sgRNAs are labeled in red and the PAM sequence in bold. (b) Genotyping of mice was performed using primers that bind outside the deleted sequence (arrows). The PCR product from a wild-type mouse (+/+) contains the exon 6 sequence while the full KO (-/-) lacks Abhd2 exon 6. Abhd2 +/– mice show a significant reduction of Abhd2 expression in both (c) qPCR and (d) western blot studies while the full KO Abhd2 –/– completely lacks expression of exon 6 and the protein. Expression levels detected by qPCR were normalized to the expression of ribosomal protein L19 ( Rpl19 ) and peptidylprolyl isomerase A ( Ppia ). Statistical significance was calculated using the unpaired t -test and the significance of changes is indicated as follows: ** p ≤ 0.01; **** p ≤ 0.0001. Abhd2 +/+ n = 4, Abhd2 +/– n = 4, and Abhd2 –/– n = 5. (e) The Abhd2 +/+ mouse ovary shows ABHD2 in the stromal cells surrounding the follicles. (f) No protein staining was detected in the Abhd2 –/– ovary.

Article Snippet: The samples were analyzed by Western blotting, using a rabbit polyclonal anti-ABHD2 antibody (1:10,000 dilution, Proteintech Group, 14039-1-AP) and a peroxidase-conjugated anti-rabbit secondary antibody (1:15,000 dilution, Abcam, ab6721).

Techniques: CRISPR, Labeling, Sequencing, Expressing, Western Blot, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle

doi: 10.3389/fcell.2021.710864

Figure Lengend Snippet: Breeding efficiency of Abhd2 –/– females.

Article Snippet: The samples were analyzed by Western blotting, using a rabbit polyclonal anti-ABHD2 antibody (1:10,000 dilution, Proteintech Group, 14039-1-AP) and a peroxidase-conjugated anti-rabbit secondary antibody (1:15,000 dilution, Abcam, ab6721).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle

doi: 10.3389/fcell.2021.710864

Figure Lengend Snippet: Abhd2 –/– female ovulation and estrous cycle. (A) Spontaneous ovulation gave rise to similar numbers of eggs for all genotypes ( n = 3 for each genotype). (B) Abhd2 ablation caused significantly increased numbers of ovulated eggs after induction of superovulation, in both 1-month-old Abhd2 +/– ( n = 8) and Abhd2 –/– ( n = 7) females compared with Abhd2 +/+ mice ( n = 8). Statistical significance was calculated using the unpaired t -test, and the significance of changes is indicated as follows: * p ≤ 0.05; *** p ≤ 0.001. (C) Age-dependent decline in the number of eggs obtained after superovulation of 1-month-old females [data from panel (B) ], as well as 7- to 8-month-old ( Abhd2 +/+ n = 5, Abhd2 +/– n = 7, and Abhd2 –/– n = 6) and 15-month-old females ( n = 3 per genotype). Representative graphs of the estrous cycle of a (D) Abhd2 +/+ and (E) Abhd2 –/– female mouse where the Abhd2 KO female shows prolonged stages in estrus although displaying cyclical changes throughout the month. (F) The combined measurements of three mice of each genotype also showed a significantly altered distribution of days in the different estrous stages, in particular, the increased percentage of days in estrus and decreased percentage of days in proestrus of Abhd2 –/– females compared with Abhd2 +/+ mice ( n = 3 for each genotype).

Article Snippet: The samples were analyzed by Western blotting, using a rabbit polyclonal anti-ABHD2 antibody (1:10,000 dilution, Proteintech Group, 14039-1-AP) and a peroxidase-conjugated anti-rabbit secondary antibody (1:15,000 dilution, Abcam, ab6721).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle

doi: 10.3389/fcell.2021.710864

Figure Lengend Snippet: Follicle count of the proestrus ovary. (a) Ovaries of 3.5-month-old Abhd2 +/– ( n = 3) and Abhd2 –/– ( n = 4) mice showed significantly lower numbers of early antral follicles, while the antral follicle number was increased compared with the Abhd2 +/+ females ( n = 4). (b) However, although hematoxylin staining of Abhd2 +/– and Abhd2 –/– ovaries displayed a higher number of atretic antral follicles compared with the wild-type ovaries, the total number of healthy follicles was similar between genotypes. (c) Representative images of Abhd2 +/+ , Abhd2 +/– , and Abhd2 –/– ovaries with apoptotic antral follicles marked by arrows. (d,e) TUNEL staining confirmed the follicles as apoptotic by labeling the granulosa cells of the atretic follicles. Statistical significance was calculated using the unpaired t -test and the significance of changes is indicated as follows: * p ≤ 0.05. Abhd2 +/+ n = 4, Abhd2 +/– n = 3, and Abhd2 –/– n = 4, for all analyses.

Article Snippet: The samples were analyzed by Western blotting, using a rabbit polyclonal anti-ABHD2 antibody (1:10,000 dilution, Proteintech Group, 14039-1-AP) and a peroxidase-conjugated anti-rabbit secondary antibody (1:15,000 dilution, Abcam, ab6721).

Techniques: Staining, TUNEL Assay, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle

doi: 10.3389/fcell.2021.710864

Figure Lengend Snippet: In vitro ovulation. Follicles collected from immature mouse ovaries ( Abhd2 +/+ n = 11 and Abhd2 –/– n = 7) were cultured to antral stage, after which ovulation was induced by incubation with hCG. (A) No difference in ovulation efficiency was observed between Abhd2 +/+ and Abhd2 –/– mice although (B) the ovaries of Abhd2 –/– mice contained a higher number of large follicles. (C) Representative image of in vitro ovulation of an Abhd2 +/+ and an Abhd2 –/– follicle. Statistical significance was calculated using the unpaired t -test and the significance of changes is indicated as follows: * p ≤ 0.05. Using 2–3 mice each time, in vitro ovulation was performed four times for Abhd2 +/+ follicles and three times for follicles collected from Abhd2 –/– mice.

Article Snippet: The samples were analyzed by Western blotting, using a rabbit polyclonal anti-ABHD2 antibody (1:10,000 dilution, Proteintech Group, 14039-1-AP) and a peroxidase-conjugated anti-rabbit secondary antibody (1:15,000 dilution, Abcam, ab6721).

Techniques: In Vitro, Cell Culture, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle

doi: 10.3389/fcell.2021.710864

Figure Lengend Snippet: Ovarian gene expression. Relative expression levels of genes involved in steroid synthesis, follicle development and ovulation were measured by qPCR using ovaries of three Abhd2 +/+ and three Abhd2 –/– 1-month old superovulated mice. (A) Expression levels were normalized to the expression of ribosomal protein L19 ( Rpl19 ) and peptidylprolyl isomerase A ( Ppia ). Follicle stimulating hormone receptor ( Fshr ), cytochrome P450 family 11 subfamily A member 1 ( Cyp11a1 ), cytochrome P450 family 17 subfamily A member 1 ( Cyp17a1 ), cytochrome P450 family 19 subfamily A member 1 ( Cyp19a1 ), vascular endothelial growth factor A ( Vegfa ), and (B) nerve growth factor ( Ngf ), neurotrophic receptor tyrosine kinase 1 ( Ntrk1 ), and nerve growth factor receptor ( Ngfr ). Statistical significance was calculated using the unpaired t -test, with p -values above columns and the significance of changes indicated as: * p ≤ 0.05.

Article Snippet: The samples were analyzed by Western blotting, using a rabbit polyclonal anti-ABHD2 antibody (1:10,000 dilution, Proteintech Group, 14039-1-AP) and a peroxidase-conjugated anti-rabbit secondary antibody (1:15,000 dilution, Abcam, ab6721).

Techniques: Gene Expression, Expressing

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Seven human ovarian serous adenocarcinoma cell lines and an immortalized human ovarian surface cell line HOSE/E7, all of which do not grow in soft agar, were used. Following transfection of the shRNA library, only OVCA420 cells formed colonies in soft agar. 43 colonies were successfully expanded. shRNAs were amplified by PCR and we reconstructed 69 different shRNA plasmids. Out of the 69 shRNAs in OVCA420 cells, 11 again generated colonies in soft agar. We then measured mRNA expression of these 11 genes using RT-PCR. Of the 11 shRNAs, shRNAs directed against ABHD2, CYB5R3 and ELAC2 suppressed target gene mRNA expression. b. Left: shRNA- ABHD2 transfected OVCA420 cell colony in soft agar. Black bar, 100 μm. Right: normalized ABHD2 / ACTB mRNA expression analyzed by RT-PCR. (n=3, respectively) c. Left: shRNA- ELAC2 transfected OVCA420 cell colony in soft agar. Right: normalized ELAC2 / ACTB mRNA expression. d. Left: shRNA- CYB5R3 transfected OVCA420 cell colony in soft agar. Right: normalized CYB5R3 / ACTB expression.

Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).

Techniques: Transfection, shRNA, Amplification, Generated, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: mRNA expression was evaluated using log2 normalized values. a. Comparison of ABHD2 mRNA expression between ovarian cancer tissues and serous borderline tumors (SBT) using gene expression microarray datasets GSE9891 and GSE2109. b. Comparison of ABHD2 mRNA expression between serous adenocarcinoma and non-serous adenocarcinoma in microarray dataset GSE2109. c. Copy number alterations for ABHD2 in TCGA samples. Del; deletion, Amp; Amplification. d. Correlation between ABHD2 copy number and mRNA expression in TCGA specimens. e. Representative ABHD2 immunohistochemistry staining for HGSOC (intensity 0, 1 and 2), normal fallopian tube and SBT are shown. Comparison of H-scores among HGSOC, fallopian tube and SBT. The H-score is calculated as 2x the percentage of the most strongly stained area plus 1x the percentage of the most weakly stained area, imparting a total score ranging from 0 to 200.

Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).

Techniques: Expressing, Microarray, Amplification, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Number of viable control, sh1-OVCA420 and sh2-OVCA420 cells following incubation on ultra-low attachment plates (n=6). b. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates. c. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction (viable cells) and Annexin V(+) fraction (apoptotic cells) between control, sh1 and sh2 cells. d. Number of viable OVCA420-control and OVCA420- ABHD2 cells following incubation on ultra-low attachment plates (n=6). Panels a-c: sh1; sh1-OVCA420, sh2; sh2-OVCA420, control; control-OVCA420; panel d: control; OVCA420-control, ABHD2 ; SKOV3- ABHD2 .

Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).

Techniques: Incubation, Staining

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Number of viable SKOV3-control and SKOV3- ABHD2 cells following incubation on ultra-low attachment plates (n=6). b. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates. c. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction and Annexin V(+) fraction between SKOV3-control and SKOV3- ABHD2 cells. d. Number of viable control- SKOV3, sh1- SKOV3- ABHD2 and sh2-SKOV3- ABHD2 cells following incubation on ultra-low attachment plates (n=6). e. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates for 48 hours. f. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction and Annexin V(+) fraction among control, sh1-SKOV3- ABHD2 and sh2-SKOV3- ABHD2 cells. Panels a-c: control ; SKOV3-control, ABHD2 ; SKOV3- ABHD2 ; panels d-f: control; control-SKOV3- ABHD2, sh1; sh1-SKOV3- ABHD2, sh2 ; sh2-SKOV3- ABHD2 .

Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).

Techniques: Incubation, Staining

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: All experiments were performed in triplicate. a. Phosphorylated p38 (P-P38) and phosphorylated ERK1/2 (P-ERK1/2) increased following knockdown of ABHD2 (sh1 and sh2) in OVCA420 cells. On the contrary, P-P38 and P-ERK1/2 decreased following overexpression of ABHD2 in SKOV3 and OVCA420 cells. b. Resistance of OVCA420 cells to anoikis on ultra-low attachment dishes was inhibited by GSK1120212, a specific inhibitor of the ERK1/2 pathway. Reduction of P-ERK1/2 following treatment with GSK1120212 was confirmed by Western blotting. DMSO, vehicle control. Cells were treated with differing doses of GSK1120212 as indicated. c. Resistance of OVCA420 cells to anoikis was inhibited following treatment with SB203580, a specific inhibitor of the the p38MAPK pathway. d. Levels of P-P38 and P-ERK1/2 increased following knockdown of ABHD2 (sh1 and sh2) in SKOV3- ABHD2 cells. e. Resistance to anoikis in sh1-SKOV3- ABHD2 and sh2-SKOV3- ABHD2 cells was inhibited following treatment with 100nM GSK1120212 (GSK) and 30μM SB203580 (SB).”

Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).

Techniques: Over Expression, Western Blot

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: ABHD2 expression levels and clinicopathological factors

Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).

Techniques: Expressing

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Differences in survival based on ABHD2 immunohistochemical scores (H-score) in HGSOC. b. Differences in survival based on ABHD2 mRNA expression in GSE9891 (n=285, mostly HGSOC) and GSE3149 (n=146, mostly HGSOC) datasets. Samples were divided into high (greater than the median value) and low (less than the median) expression cases. c. Analysis of HGSOC patients (n=36) from KOV-75 based on ABHD2 mRNA expression. d. Analysis of non-HGSOC patients (n=39) from KOV-75 based on ABHD2 mRNA expression. n.s., not significant.

Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).

Techniques: Immunohistochemical staining, Expressing

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Representative data showing 7-ADD staining following 24 hour incubation with 10 μM cisplatin. b. The ratio of the 7-AAD negative live OVCA420 cells markedly increased following suppression of ABHD2 (sh1 and sh2) as compared to the control after 24 hour incubation with 10 μM cisplatin (n=3). c. Dose-response curves following incubation of OVCA420 cells with the indicated concentrations of cisplatin for 72 hours (n=6). d. Cisplatin IC50 values increased following suppression of ABHD2 in OVCA420 cells. e. Representative data showing 7-ADD staining following 24 hour incubation with 100 μM Carboplatin. f. The ratio of 7-AAD negative live OVCA420 cells markedly increased following suppression of ABHD2 (sh1 and sh2) as compared to the control following a 24 hour incubation with 100 μM carboplatin (n=3).

Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).

Techniques: Staining, Incubation

Journal: Journal of Proteome Research

Article Title: Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics

doi: 10.1021/acs.jproteome.3c00641

Figure Lengend Snippet: (A) Protein and (B) mRNA levels of ABHD2 and ADAM15 in parental, mock, and Casp9 KO MC3T3-E1 cells; α-tubulin was used as a loading control for immunoblotting. Data represents means ± SD from at least three independent experiments.

Article Snippet: Blots were probed with CASP-9 (#9508, Cell Signaling Technology, USA), cleaved CASP-3 (#9661, Cell Signaling Technology, USA), ABHD2 (14039-1-AP, Proteintech, Germany), ADAM15 (GTX101599, GeneTex, USA), BST-2 (sc-390719, Santa Cruz Biotechnology, USA; 13560-1-AP, Proteintech, Germany; #60066S, Cell Signaling Technology, USA), or α-tubulin (ab7291, Abcam, UK) specific antibodies and horseradish peroxidase-conjugated mouse or rabbit secondary antibodies (Sigma-Aldrich, USA).

Techniques: Control, Western Blot

Journal: Journal of Proteome Research

Article Title: Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics

doi: 10.1021/acs.jproteome.3c00641

Figure Lengend Snippet: Protein levels of ABHD2 and ADAM15 in MC3T3-E1 cells with inhibited CASP-9 or CASP-3/-7; α-tubulin was used as a loading control.

Article Snippet: Blots were probed with CASP-9 (#9508, Cell Signaling Technology, USA), cleaved CASP-3 (#9661, Cell Signaling Technology, USA), ABHD2 (14039-1-AP, Proteintech, Germany), ADAM15 (GTX101599, GeneTex, USA), BST-2 (sc-390719, Santa Cruz Biotechnology, USA; 13560-1-AP, Proteintech, Germany; #60066S, Cell Signaling Technology, USA), or α-tubulin (ab7291, Abcam, UK) specific antibodies and horseradish peroxidase-conjugated mouse or rabbit secondary antibodies (Sigma-Aldrich, USA).

Techniques: Control

Journal: Journal of Proteome Research

Article Title: Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics

doi: 10.1021/acs.jproteome.3c00641

Figure Lengend Snippet: Proteins Acting as Negative Regulators of Cell Migration Upregulated after Casp9 KO in Both Clones Compared to Mock and wt Cells a

Article Snippet: Blots were probed with CASP-9 (#9508, Cell Signaling Technology, USA), cleaved CASP-3 (#9661, Cell Signaling Technology, USA), ABHD2 (14039-1-AP, Proteintech, Germany), ADAM15 (GTX101599, GeneTex, USA), BST-2 (sc-390719, Santa Cruz Biotechnology, USA; 13560-1-AP, Proteintech, Germany; #60066S, Cell Signaling Technology, USA), or α-tubulin (ab7291, Abcam, UK) specific antibodies and horseradish peroxidase-conjugated mouse or rabbit secondary antibodies (Sigma-Aldrich, USA).

Techniques: Migration, Clone Assay