dpp3 rabbit polyclonal  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: The serine protease DPP9 and the redox sensor KEAP1 form a mutually inhibitory complex

doi: 10.1016/j.jbc.2024.108034

Figure Lengend Snippet: KEAP1-bound DPP9 is catalytically inactive. A , FLAG-tagged DPP9 was expressed alone, and V5-tagged DPP9 was co-expressed with FLAG-tagged FL KEAP1 or the isolated KELCH domain in DPP8/9 −/− HEK 293T cells. The resulting lysates were subjected to anti-FLAG IP, and the protein levels and DPP9 activity in the eluates were assessed by immunoblotting and GP-AMC reporter assays, respectively. The DPP9-FLAG eluate was assessed in a five-point, two-fold dilution series to normalize the free DPP9 protein levels to the KEAP1-bound DPP9 protein levels ( red arrows indicate comparable levels). B–F , the indicated WT and mutant proteins were transiently expressed in DPP8/9 −/− ( B–D , and F ) or WT ( E ) HEK 293T cells. KI696 (5 μΜ) was added to the indicated samples at the time of transfection. After 24 h, DPP9 and DPP3 activity in intact cells and/or harvested lysates (as indicated) was assessed using GP-AMC and RR-AMC assays, respectively. Harvested lysates were also analyzed by immunoblotting. GP-AMC data are means ± SEM. All data, including immunoblots, are representative of three or more independent experiments.

Article Snippet: Antibodies used include: DPP9 rabbit polyclonal antibody (Abcam, ab42080), KEAP1 rabbit polyclonal antibody (Proteintech, 10503-2-AP), Myc-Tag rabbit monoclonal antibody (71D1O, Cell Signaling Technology), NRF2 rabbit polyclonal antibody (Abcam, ab137550), DPP3 rabbit polyclonal (Proteintech, 10650-1-AP), β-Tubulin rabbit monoclonal antibody (Cell Signaling, 2128), Lamin B1 rabbit polyclonal antibody (Proteintech, 12987-1-AP), FLAG mouse monoclonal Ab (Sigma Aldrich, F1804), HA mouse monoclonal Ab (Cell Signaling Tech, 6E2), V5 mouse monoclonal antibody (Sigma, V8012), IRDye 680RD Streptavidin (LICOR, 926-68079) IRDye 800CW donkey anti-rabbit (LICOR, 925-32211), IRDye 680RD donkey anti-rabbit (925-68073), IRDye 800CW donkey anti-mouse (925-32212), IRDye 680RD donkey anti-mouse (925-68072).

Techniques: Isolation, Activity Assay, Western Blot, Mutagenesis, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The serine protease DPP9 and the redox sensor KEAP1 form a mutually inhibitory complex

doi: 10.1016/j.jbc.2024.108034

Figure Lengend Snippet: C844 on DPP9 is required for KEAP1 binding . A , overlay of the AlphaFold 3 predictions of the DPP9, NRF2, and DPP3 E(S/T)GE peptides. B , sequence alignment of the peptides in A . C–G , the indicated proteins were transiently expressed in DPP8/9 −/− HEK 293T cells. The lysates and anti-FLAG IPs were analyzed by immunoblotting (SDS- and BN-PAGE) and GP-AMC assays as indicated. GP-AMC data are means ± SEM. All data, including immunoblots, are representative of three or more independent experiments.

Article Snippet: Antibodies used include: DPP9 rabbit polyclonal antibody (Abcam, ab42080), KEAP1 rabbit polyclonal antibody (Proteintech, 10503-2-AP), Myc-Tag rabbit monoclonal antibody (71D1O, Cell Signaling Technology), NRF2 rabbit polyclonal antibody (Abcam, ab137550), DPP3 rabbit polyclonal (Proteintech, 10650-1-AP), β-Tubulin rabbit monoclonal antibody (Cell Signaling, 2128), Lamin B1 rabbit polyclonal antibody (Proteintech, 12987-1-AP), FLAG mouse monoclonal Ab (Sigma Aldrich, F1804), HA mouse monoclonal Ab (Cell Signaling Tech, 6E2), V5 mouse monoclonal antibody (Sigma, V8012), IRDye 680RD Streptavidin (LICOR, 926-68079) IRDye 800CW donkey anti-rabbit (LICOR, 925-32211), IRDye 680RD donkey anti-rabbit (925-68073), IRDye 800CW donkey anti-mouse (925-32212), IRDye 680RD donkey anti-mouse (925-68072).

Techniques: Binding Assay, Sequencing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: Generation and validation of DPP3 knockout mice. A , strategy for the generation of DPP3 -knockout mice. B and C , demonstration of the lack of DPP3 in male DPP3 −/− mice by Western blotting ( B ) and activity assays using Arg-Arg-2-naphthylamide as an artificial substrate ( C ). BAT , brown adipose tissue; SI , small intestine; SM , skeletal muscle; WAT , white adipose tissue (12–16 weeks of age; n = 3/group); n.d. , non detectable. **, p < 0.01; ***, p < 0.001 versus WT mice based on unpaired two-sided Student's t test. The data are representative of three technical replicates from three biological replicates and presented as means ± S.D.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques: Biomarker Discovery, Knock-Out, Western Blot, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: DPP3 −/− mice display lower body weight and altered food and water intake. A and B , body weight ( A ) and body composition ( B ) of male mice fed a regular chow diet. C–H , cumulative ( top panels ) and total ( bottom panels ) food intake ( C and F , respectively), water consumption ( D and G , respectively), and locomotive motion ( E and H , respectively) were measured in metabolic cages over the light and dark phases in male DPP3 −/− and DPP3 +/+ mice (12–16 weeks of age; n = 6/group) fed a regular chow diet over a period of 6 consecutive days. *, p < 0.05; **, p < 0.01 versus WT mice based on unpaired two-sided Student's t test. The data are representative for two independent cohorts and presented as the means ± S.D.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: DPP3 knockout mice display unaltered energy expenditure. Cumulative and daily oxygen consumption ( A and D , respectively), carbon-dioxide production ( B and E , respectively), respiratory exchange ratio ( RER , C ), and EE ( F ) were measured in metabolic cages over the light and dark phases in male DPP3 −/− and DPP3 +/+ mice (12–16 weeks of age; n = 6/group) fed a regular chow diet over a period of 6 consecutive days. *, p < 0.05; **, p < 0.01 versus WT mice based on unpaired two-sided Student's t test. The data are representative of two independent cohorts and presented as the means ± S.D.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques: Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: DPP3 −/− mice exhibit significantly elevated water consumption during fasting. A and B , cumulative ( A ) and daily ( B ) water intake during a 13-h fasting period was measured in metabolic cages in male DPP3 −/− and DPP3 +/+ mice (12–16 weeks of age; n = 6/group). *, p < 0.05; **, p < 0.01 versus WT mice based on unpaired two-sided Student's t test. The data represent the means ± S.D.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: DPP3 −/− mice exhibit increased circulating angiotensin metabolites. A–C , concentration of RAS peptides ( A ), aldosterone ( B ), and renin ( C ) activity in serum measured by LC–MS in male DPP3 −/− and DPP3 +/+ mice (12–16 weeks of age; n = 8/group). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus WT mice based on unpaired two-sided Student's t test. The data represent the means ± S.D.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques: Concentration Assay, Activity Assay, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: DPP3 knockout renders mice susceptible to oxidative stress. A–C , quantification of ( ROS production ( A ), lipid peroxidation ( B ), and catalase activity ( C ) in kidney homogenates of male DPP3 −/− and DPP3 +/+ mice (12–16 weeks of age; n = 5/group). *, p < 0.05; **, p < 0.01 versus WT mice based on unpaired two-sided Student's t test. The experiments were performed in technical triplicates, and the data represent the means ± S.D.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques: Knock-Out, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: DPP3 deletion does not affect morphology of mouse kidney. Periodic acid–Schiff–stained slides of renal histology showing unremarkable glomeruli and tubuli in DPP3 −/− ( A ) and DPP3 +/+ ( B ) mice. Left panels , 10×; right panels , 20×.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: DPP3 deficiency does not lead to changes in blood pressure. Systolic and diastolic blood pressure were measured by tail-cuff method in male DPP3 +/+ and DPP3 −/− mice (18–22 weeks of age; n = 8/group). The data are presented as the means ± S.D.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: RAS peptides can be “good” or “bad” substrates of DPP3. RAS peptides turned over by DPP3 ( DP ), thus acting as “good” substrates ( A–C ). Raw data showing the heat change of the reaction as a function of time ( top panels ) and fitted curve for the rate of reaction ( bottom panels ) of Ang II ( A ), Ang(1–7) ( B ), and Ang(1–5) ( C ). Some RAS peptides demonstrated both endothermic and exothermic behavior, thus acting as “slow” substrates of DPP3 ( D–F ). The biphasic peaks were most likely due to binding to DPP3 and a subsequent slow turnover event. Raw data showing the heat change of the reaction in the case of slow substrates, tynorphin ( D ), Ang III ( E ), and Ang IV ( F ). Curve fitting was not possible in the case of slow substrates. The reaction was started by injecting 5 µl of 2 m m angiotensin peptides to the calorimetric cell containing 20 μ m hDPP3. The data represent three or more technical replicates from two biological replicates.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice

doi: 10.1074/jbc.RA120.014183

Figure Lengend Snippet: DPP3 exerts a sex-specific effect on the knockout mice. A , body weight ( A ) and body composition of female mice ( B ) fed a regular chow diet. C and D , daily food intake ( C ) and water consumption ( D ) was measured in metabolic cages over the light and dark phases in DPP3 −/− and DPP3 +/+ female mice (12–16 weeks of age; n = 6/group) fed a regular chow diet over a period of 6 consecutive days. E , concentration of RAS peptides in serum measured by LC–MS in DPP3 −/− and DPP3 +/+ female mice (12–16 weeks of age; n = 8/group). F , quantification of ROS production in DPP3 −/− and DPP3 +/+ female mice (12–16 weeks of age; n = 5/group). *, p < 0.05; **, p < 0.01 versus WT mice based on unpaired two-sided Student's t test. The data represent means ± S.D.

Article Snippet: The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C.

Techniques: Knock-Out, Concentration Assay, Liquid Chromatography with Mass Spectroscopy