Journal: bioRxiv

Article Title: ABHD2 activity is not required for the non-genomic action of progesterone on human sperm

doi: 10.1101/2024.12.17.628646

Figure Lengend Snippet: Characterisation of ABHD2 inhibitors. (A) structures of the inhibitors with enzymatic IC50 values and T M shifts obtained with ABHD2(L33-E425). (B) representative inhibition curves. (C) representative melting and derivative curves

Article Snippet: For expression in insect cells, a codon optimized insert encoding for full-length human ABHD2 was synthesized at Thermofisher Scientific and cloned via Gateway technology (Invitrogen) into pD-INS26, a vector backbone produced in-house by modification of pVL1393 to encode for a C-terminal eGFP-6xHis tag.

Techniques: Inhibition

Journal: bioRxiv

Article Title: ABHD2 activity is not required for the non-genomic action of progesterone on human sperm

doi: 10.1101/2024.12.17.628646

Figure Lengend Snippet: Endogenous steroids (5 µM) do not modulate the enzymatic activity (A) nor lead to thermal stabilization (B) of ABHD2(L22-E425). A. Enzymatic activity. B. DSF curves of representative compounds

Article Snippet: For expression in insect cells, a codon optimized insert encoding for full-length human ABHD2 was synthesized at Thermofisher Scientific and cloned via Gateway technology (Invitrogen) into pD-INS26, a vector backbone produced in-house by modification of pVL1393 to encode for a C-terminal eGFP-6xHis tag.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: ABHD2 activity is not required for the non-genomic action of progesterone on human sperm

doi: 10.1101/2024.12.17.628646

Figure Lengend Snippet: Thermostabilization of ABHD2 by inhibitors in intact cells. A. Cells were incubated with compound 1, compound 6 (171) or DMSO followed by a 3 minute incubation at the indicated temperatures. Remaining ABHD2 protein levels were monitored by lumincescence and normalized to cells incubated at 37°C. B. Cells incubated with the indicated compounds, then heated for 3 minutes at 50°C. The remaining ABHD2 was measured and normalized as in A.

Article Snippet: For expression in insect cells, a codon optimized insert encoding for full-length human ABHD2 was synthesized at Thermofisher Scientific and cloned via Gateway technology (Invitrogen) into pD-INS26, a vector backbone produced in-house by modification of pVL1393 to encode for a C-terminal eGFP-6xHis tag.

Techniques: 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: Consecutive sections were pretreated in citrate buffer (10 min/98 °C) for antigen retrieval and then incubated with ABHD2 antibody (14039-1-AP, Proteintech, Germany) or antibody specific to cleaved CASP-9 (9509, Cell Signaling Technology, USA) overnight.

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: Consecutive sections were pretreated in citrate buffer (10 min/98 °C) for antigen retrieval and then incubated with ABHD2 antibody (14039-1-AP, Proteintech, Germany) or antibody specific to cleaved CASP-9 (9509, Cell Signaling Technology, USA) overnight.

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: Immunofluorescent staining of ABHD2 (A, C) and cleaved caspase 9 (clCASP9) (B, D) in the developing mouse frontal limb at prenatal day E15. Both proteins were detected in consecutive sections. Positive signal in green (arrows), nuclei counterstained with DAPI (blue); total magnification ×20 (A, B) or ×40 (C, D). hc, hypertrophic cartilage.

Article Snippet: Consecutive sections were pretreated in citrate buffer (10 min/98 °C) for antigen retrieval and then incubated with ABHD2 antibody (14039-1-AP, Proteintech, Germany) or antibody specific to cleaved CASP-9 (9509, Cell Signaling Technology, USA) overnight.

Techniques: Staining

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: Consecutive sections were pretreated in citrate buffer (10 min/98 °C) for antigen retrieval and then incubated with ABHD2 antibody (14039-1-AP, Proteintech, Germany) or antibody specific to cleaved CASP-9 (9509, Cell Signaling Technology, USA) overnight.

Techniques: Migration, Clone Assay

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: Figure 5. (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: Cells were lysed, and proteins were resolved by SDS-PAGE and immunoblotted as described previously.46 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: Figure 6. 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: Cells were lysed, and proteins were resolved by SDS-PAGE and immunoblotted as described previously.46 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: Figure 7. Immunofluorescent staining of ABHD2 (A, C) and cleaved caspase 9 (clCASP9) (B, D) in the developing mouse frontal limb at prenatal day E15. Both proteins were detected in consecutive sections. Positive signal in green (arrows), nuclei counterstained with DAPI (blue); total magnification ×20 (A, B) or ×40 (C, D). hc, hypertrophic cartilage.

Article Snippet: Cells were lysed, and proteins were resolved by SDS-PAGE and immunoblotted as described previously.46 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: Staining