Journal: Neurobiology of aging

Article Title: Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain

doi: 10.1016/j.neurobiolaging.2018.10.027

Figure Lengend Snippet: List of antibodies used

Article Snippet: A detailed summary of all of the antibodies used in these studies is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Phospho-Epitope Type Source Manufacturer Location Catalog # WB Dilution Tau Tau46 Total Tau Monoclonal Mouse Cell Signaling Danvers, MA 4019 1:2500 Anti-Human Tau A0024 Total Tau 243-441 Polyclonal Rabbit Dako Cytomation Carpinteria, CA A002401-2 1:10000 TG5 Total Tau 220-240 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT8 pSer202/pThr205 Monoclonal Mouse Thermo Scientific Waltham, MA MN1020 1:10000 CP13 pSer202 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 PHF-1 pSer396/pSer404 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT180 pThr231 Monoclonal Mouse Thermo Scientific Waltham, MA MN1040 1:1000 PS422 pSer422 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-764G 1:1000 PS262 pSer262 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-750G 1:1000 PS199 pSer199 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-734G 1:1000 AT270 pThr181 Monoclonal Mouse Thermo Scientific Waltham, MA MN1050 1:1000 12 E8 pSer262/pSer356 Monoclonal Mouse Pharmaceuticals Francisco, CA N/A 1:1000 Kinases GSK-3β Monoclonal Rabbit Cell Signaling Danvers, MA 9315 1:1000 Phospho-GSK-3 β (Ser9) Ser9 Polyclonal Rabbit Cell Signaling Danvers, MA 9336 1:1000 SAPK/JNK Monoclonal Rabbit Cell Signaling Danvers, MA 9258 1:1000 Phospho-SAPK/JNK Thr183/Tyr185 Monoclonal Rabbit Cell Signaling Danvers, MA 4671 1:1000 p44/42 MAPK Monoclonal Mouse Cell Signaling Danvers, MA 9107 1:2000 Phospho-p44/42 MAPK Thr202/Tyr204 Monoclonal Mouse Cell Signaling Danvers, MA 9106 1:2000 CaMKII Polyclonal Rabbit Cell Signaling Danvers, MA 3362 1:1000 Phospho-CaMKII Thr286 Polyclonal Rabbit Cell Signaling Danvers, MA 3361 1:1000 p35/25 Monoclonal Rabbit Cell Signaling Danvers, MA 2680 1:1000 Phosphatases PP2A-C (catalytic subunit) Monoclonal Mouse Sigma-Aldrich St. Louis, MO A5316 1:10000 demethylated PP2A-C Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 PP1 (E-9) Monoclonal Mouse Santa Cruz Dallas, TX sc-7482 1:1000 Pan-Calcineurin A (PP2B) Polyclonal Rabbit Cell Signaling Danvers, MA 2614 1:1000 PP5 Polyclonal Rabbit Cell Signaling Danvers, MA 2289 1:1000 Gel loading Controls β-actin Monoclonal Mouse Sigma-Aldrich St.Louis, MO A5316 1:10000 β3-tubulin Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 Open in a separate window List of antibodies used 2.3.

Techniques:

Journal: Neurobiology of aging

Article Title: Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain

doi: 10.1016/j.neurobiolaging.2018.10.027

Figure Lengend Snippet: 1. Hippocampal tau phosphorylation (% Ctl) at the AT8 (pSer202/pThr205), CP13 (pSer202), and PHF-1 (pSer396/Ser404) phosphoepitopes (1A-C) in 8-10-week-old C57BL/6 treated with midazolam (Mid) 10 mg/kg (n = 5) or 25 mg/kg (n = 5) i.p. Control (Ctl) mice (n = 4) were treated with 0.9% saline (100 μL per 10 g of body weight). All mice were sacrificed 30 min after treatment. No statistically significant difference in rectal temperatures (°C) was observed at the time of tissue harvest: Ctl 37.4 ± 0.3°C, Mid 10 mg/kg 37.3 ± 0.3°C, and Mid 25 mg/kg 37.4 ± 0.2°C. 2. Hippocampal tau phosphorylation (% Ctl) at the AT8, CP13, and PHF-1 phosphoepitopes (2A-C) in 8-10-week-old C57BL/6 mice treated with midazolam 25 mg/kg (MID 6h, n = 5) or 0.9% saline (Ctl, n = 4) i.p. and sacrificed 6h later. Normothermia was maintained throughout the study and rectal temperatures were similar at the time of hippocampal tissue harvest: Ctl 36.4 ± 0.3°C and MID 6h 36.9 ± 0.2 °C. Phosphorylated tau was normalized to total tau (1D and 2D) after controlling for gel loading with β-actin. Relative immunoreactive band intensities are expressed as a percent of Ctl and are displayed for each phosphoepitope and total tau. For each condition, 2 representative immunoblot bands are displayed. Data are expressed as mean ± SD and were analyzed using ANOVA with Tukey’s post-hoc test. * and ** denote P < 0.05 and P < 0.01 vs. Ctl, respectively.

Article Snippet: A detailed summary of all of the antibodies used in these studies is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Phospho-Epitope Type Source Manufacturer Location Catalog # WB Dilution Tau Tau46 Total Tau Monoclonal Mouse Cell Signaling Danvers, MA 4019 1:2500 Anti-Human Tau A0024 Total Tau 243-441 Polyclonal Rabbit Dako Cytomation Carpinteria, CA A002401-2 1:10000 TG5 Total Tau 220-240 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT8 pSer202/pThr205 Monoclonal Mouse Thermo Scientific Waltham, MA MN1020 1:10000 CP13 pSer202 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 PHF-1 pSer396/pSer404 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT180 pThr231 Monoclonal Mouse Thermo Scientific Waltham, MA MN1040 1:1000 PS422 pSer422 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-764G 1:1000 PS262 pSer262 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-750G 1:1000 PS199 pSer199 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-734G 1:1000 AT270 pThr181 Monoclonal Mouse Thermo Scientific Waltham, MA MN1050 1:1000 12 E8 pSer262/pSer356 Monoclonal Mouse Pharmaceuticals Francisco, CA N/A 1:1000 Kinases GSK-3β Monoclonal Rabbit Cell Signaling Danvers, MA 9315 1:1000 Phospho-GSK-3 β (Ser9) Ser9 Polyclonal Rabbit Cell Signaling Danvers, MA 9336 1:1000 SAPK/JNK Monoclonal Rabbit Cell Signaling Danvers, MA 9258 1:1000 Phospho-SAPK/JNK Thr183/Tyr185 Monoclonal Rabbit Cell Signaling Danvers, MA 4671 1:1000 p44/42 MAPK Monoclonal Mouse Cell Signaling Danvers, MA 9107 1:2000 Phospho-p44/42 MAPK Thr202/Tyr204 Monoclonal Mouse Cell Signaling Danvers, MA 9106 1:2000 CaMKII Polyclonal Rabbit Cell Signaling Danvers, MA 3362 1:1000 Phospho-CaMKII Thr286 Polyclonal Rabbit Cell Signaling Danvers, MA 3361 1:1000 p35/25 Monoclonal Rabbit Cell Signaling Danvers, MA 2680 1:1000 Phosphatases PP2A-C (catalytic subunit) Monoclonal Mouse Sigma-Aldrich St. Louis, MO A5316 1:10000 demethylated PP2A-C Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 PP1 (E-9) Monoclonal Mouse Santa Cruz Dallas, TX sc-7482 1:1000 Pan-Calcineurin A (PP2B) Polyclonal Rabbit Cell Signaling Danvers, MA 2614 1:1000 PP5 Polyclonal Rabbit Cell Signaling Danvers, MA 2289 1:1000 Gel loading Controls β-actin Monoclonal Mouse Sigma-Aldrich St.Louis, MO A5316 1:10000 β3-tubulin Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 Open in a separate window List of antibodies used 2.3.

Techniques: Phospho-proteomics, Control, Saline, Western Blot

Journal: Neurobiology of aging

Article Title: Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain

doi: 10.1016/j.neurobiolaging.2018.10.027

Figure Lengend Snippet: Cortical tau phosphorylation (% Ctl) at the AT8 (A), CP13 (B), PHF-1 (C), 12E8 (D), AT180 (E), AT270 (F) and pS199 (G) phosphoepitopes in 8-10-week old C57BL/6 mice treated with midazolam (Mid) 25 mg/kg i.p and sacrificed 30 min (0.5h, n = 6) or 24h (n = 6) later. Control mice (n = 6) were treated with 0.9% saline i.p. and sacrificed 30 min later. Normothermia was maintained throughout and rectal temperatures at the end of the study were 37.4 ± 0.2°C (Ctl), 37.0 ± 0.3°C (Mid 0.5h), and 37.0 ± 0.4°C (Mid 24h). Phosphorylated tau levels were normalized to total tau (H) after controlling for gel loading with β-actin. Data are expressed as mean ± SD and **,***, **** denote P < 0.01, P <0.001, and P < 0.0001 vs. Ctl., respectively using ANOVA with Tukey’s post-hoc test. Two representative immunoblot bands are displayed for each condition.

Article Snippet: A detailed summary of all of the antibodies used in these studies is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Phospho-Epitope Type Source Manufacturer Location Catalog # WB Dilution Tau Tau46 Total Tau Monoclonal Mouse Cell Signaling Danvers, MA 4019 1:2500 Anti-Human Tau A0024 Total Tau 243-441 Polyclonal Rabbit Dako Cytomation Carpinteria, CA A002401-2 1:10000 TG5 Total Tau 220-240 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT8 pSer202/pThr205 Monoclonal Mouse Thermo Scientific Waltham, MA MN1020 1:10000 CP13 pSer202 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 PHF-1 pSer396/pSer404 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT180 pThr231 Monoclonal Mouse Thermo Scientific Waltham, MA MN1040 1:1000 PS422 pSer422 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-764G 1:1000 PS262 pSer262 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-750G 1:1000 PS199 pSer199 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-734G 1:1000 AT270 pThr181 Monoclonal Mouse Thermo Scientific Waltham, MA MN1050 1:1000 12 E8 pSer262/pSer356 Monoclonal Mouse Pharmaceuticals Francisco, CA N/A 1:1000 Kinases GSK-3β Monoclonal Rabbit Cell Signaling Danvers, MA 9315 1:1000 Phospho-GSK-3 β (Ser9) Ser9 Polyclonal Rabbit Cell Signaling Danvers, MA 9336 1:1000 SAPK/JNK Monoclonal Rabbit Cell Signaling Danvers, MA 9258 1:1000 Phospho-SAPK/JNK Thr183/Tyr185 Monoclonal Rabbit Cell Signaling Danvers, MA 4671 1:1000 p44/42 MAPK Monoclonal Mouse Cell Signaling Danvers, MA 9107 1:2000 Phospho-p44/42 MAPK Thr202/Tyr204 Monoclonal Mouse Cell Signaling Danvers, MA 9106 1:2000 CaMKII Polyclonal Rabbit Cell Signaling Danvers, MA 3362 1:1000 Phospho-CaMKII Thr286 Polyclonal Rabbit Cell Signaling Danvers, MA 3361 1:1000 p35/25 Monoclonal Rabbit Cell Signaling Danvers, MA 2680 1:1000 Phosphatases PP2A-C (catalytic subunit) Monoclonal Mouse Sigma-Aldrich St. Louis, MO A5316 1:10000 demethylated PP2A-C Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 PP1 (E-9) Monoclonal Mouse Santa Cruz Dallas, TX sc-7482 1:1000 Pan-Calcineurin A (PP2B) Polyclonal Rabbit Cell Signaling Danvers, MA 2614 1:1000 PP5 Polyclonal Rabbit Cell Signaling Danvers, MA 2289 1:1000 Gel loading Controls β-actin Monoclonal Mouse Sigma-Aldrich St.Louis, MO A5316 1:10000 β3-tubulin Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 Open in a separate window List of antibodies used 2.3.

Techniques: Phospho-proteomics, Control, Saline, Western Blot

Journal: Neurobiology of aging

Article Title: Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain

doi: 10.1016/j.neurobiolaging.2018.10.027

Figure Lengend Snippet: Hippocampal tau phosphorylation (% Ctl) at the AT8, CP13, and PHF-1 phosphoepitopes (1A-C) in 3-month-old hTau mice treated with midazolam 25 mg/kg (n = 6) or saline (Ctl, n = 6) i.p. and sacrificed 6h later. Normothermia was maintained throughout the study and rectal temperatures were similar at the time of hippocampal tissue harvest: Ctl 36.8 ± 0.9°C and Midazolam 36.8 ± 0.7 °C. Phosphorylated tau levels were normalized to total tau (1D), after controlling for gel loading with β-actin. Levels of tau in the total (2A), sarkosyl-insoluble (2B), sand heat stabilized fractions (2C) were measured using immunoblotting and total tau antibody. Relative immunoreactive band intensities are expressed as a percent of Ctl and are displayed for each phosphoepitope and total tau. For each condition, 2 representative immunoblot bands are displayed. Data are expressed as mean ± SD and *** P < 0.001 vs. Ctl using an unpaired t-test.

Article Snippet: A detailed summary of all of the antibodies used in these studies is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Phospho-Epitope Type Source Manufacturer Location Catalog # WB Dilution Tau Tau46 Total Tau Monoclonal Mouse Cell Signaling Danvers, MA 4019 1:2500 Anti-Human Tau A0024 Total Tau 243-441 Polyclonal Rabbit Dako Cytomation Carpinteria, CA A002401-2 1:10000 TG5 Total Tau 220-240 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT8 pSer202/pThr205 Monoclonal Mouse Thermo Scientific Waltham, MA MN1020 1:10000 CP13 pSer202 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 PHF-1 pSer396/pSer404 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT180 pThr231 Monoclonal Mouse Thermo Scientific Waltham, MA MN1040 1:1000 PS422 pSer422 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-764G 1:1000 PS262 pSer262 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-750G 1:1000 PS199 pSer199 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-734G 1:1000 AT270 pThr181 Monoclonal Mouse Thermo Scientific Waltham, MA MN1050 1:1000 12 E8 pSer262/pSer356 Monoclonal Mouse Pharmaceuticals Francisco, CA N/A 1:1000 Kinases GSK-3β Monoclonal Rabbit Cell Signaling Danvers, MA 9315 1:1000 Phospho-GSK-3 β (Ser9) Ser9 Polyclonal Rabbit Cell Signaling Danvers, MA 9336 1:1000 SAPK/JNK Monoclonal Rabbit Cell Signaling Danvers, MA 9258 1:1000 Phospho-SAPK/JNK Thr183/Tyr185 Monoclonal Rabbit Cell Signaling Danvers, MA 4671 1:1000 p44/42 MAPK Monoclonal Mouse Cell Signaling Danvers, MA 9107 1:2000 Phospho-p44/42 MAPK Thr202/Tyr204 Monoclonal Mouse Cell Signaling Danvers, MA 9106 1:2000 CaMKII Polyclonal Rabbit Cell Signaling Danvers, MA 3362 1:1000 Phospho-CaMKII Thr286 Polyclonal Rabbit Cell Signaling Danvers, MA 3361 1:1000 p35/25 Monoclonal Rabbit Cell Signaling Danvers, MA 2680 1:1000 Phosphatases PP2A-C (catalytic subunit) Monoclonal Mouse Sigma-Aldrich St. Louis, MO A5316 1:10000 demethylated PP2A-C Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 PP1 (E-9) Monoclonal Mouse Santa Cruz Dallas, TX sc-7482 1:1000 Pan-Calcineurin A (PP2B) Polyclonal Rabbit Cell Signaling Danvers, MA 2614 1:1000 PP5 Polyclonal Rabbit Cell Signaling Danvers, MA 2289 1:1000 Gel loading Controls β-actin Monoclonal Mouse Sigma-Aldrich St.Louis, MO A5316 1:10000 β3-tubulin Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 Open in a separate window List of antibodies used 2.3.

Techniques: Phospho-proteomics, Saline, Western Blot

Journal: Neurobiology of aging

Article Title: Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain

doi: 10.1016/j.neurobiolaging.2018.10.027

Figure Lengend Snippet: Hippocampal tau phosphorylation (% Ctl) at the AT8, CP13, PHF-1, AT180, AT270, pSer199, pSer262 phosphoepitopes in 8-10-week-old (A-G) or 11-month-old (I-O) C57BL/6 mice treated with a subcutaneous infusion of midazolam 3 mg/kg/h (n = 6 young; n = 5 old) or 0.9% saline (Ctl) (n = 6 young; n = 5 old) i.p. and sacrificed 24h after the start of the infusion. Normothermia was maintained throughout the administration of midazolam or saline, and rectal temperatures were similar at the time of hippocampal tissue harvest: young Ctl 36.5 ± 0.2°C and Midazolam 36.8 ± 0.8 °C and old Ctl 36.9 ± 0.4°C and Midazolam 36.5 ± 0.04 °C. Phosphorylated tau levels were normalized to each groups respective level of total tau (H,P), after controlling for gel loading with β-actin. Relative immunoreactive band intensities are expressed as a percent of Ctl and are displayed for each phosphoepitope and total tau. For each condition, 2 representative immunoblot bands are displayed. Data are expressed as mean ± SD and ***, **** denote P < 0.001 and P < 0.0001, respectively, vs. Ctl using an unpaired t-test.

Article Snippet: A detailed summary of all of the antibodies used in these studies is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Phospho-Epitope Type Source Manufacturer Location Catalog # WB Dilution Tau Tau46 Total Tau Monoclonal Mouse Cell Signaling Danvers, MA 4019 1:2500 Anti-Human Tau A0024 Total Tau 243-441 Polyclonal Rabbit Dako Cytomation Carpinteria, CA A002401-2 1:10000 TG5 Total Tau 220-240 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT8 pSer202/pThr205 Monoclonal Mouse Thermo Scientific Waltham, MA MN1020 1:10000 CP13 pSer202 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 PHF-1 pSer396/pSer404 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT180 pThr231 Monoclonal Mouse Thermo Scientific Waltham, MA MN1040 1:1000 PS422 pSer422 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-764G 1:1000 PS262 pSer262 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-750G 1:1000 PS199 pSer199 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-734G 1:1000 AT270 pThr181 Monoclonal Mouse Thermo Scientific Waltham, MA MN1050 1:1000 12 E8 pSer262/pSer356 Monoclonal Mouse Pharmaceuticals Francisco, CA N/A 1:1000 Kinases GSK-3β Monoclonal Rabbit Cell Signaling Danvers, MA 9315 1:1000 Phospho-GSK-3 β (Ser9) Ser9 Polyclonal Rabbit Cell Signaling Danvers, MA 9336 1:1000 SAPK/JNK Monoclonal Rabbit Cell Signaling Danvers, MA 9258 1:1000 Phospho-SAPK/JNK Thr183/Tyr185 Monoclonal Rabbit Cell Signaling Danvers, MA 4671 1:1000 p44/42 MAPK Monoclonal Mouse Cell Signaling Danvers, MA 9107 1:2000 Phospho-p44/42 MAPK Thr202/Tyr204 Monoclonal Mouse Cell Signaling Danvers, MA 9106 1:2000 CaMKII Polyclonal Rabbit Cell Signaling Danvers, MA 3362 1:1000 Phospho-CaMKII Thr286 Polyclonal Rabbit Cell Signaling Danvers, MA 3361 1:1000 p35/25 Monoclonal Rabbit Cell Signaling Danvers, MA 2680 1:1000 Phosphatases PP2A-C (catalytic subunit) Monoclonal Mouse Sigma-Aldrich St. Louis, MO A5316 1:10000 demethylated PP2A-C Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 PP1 (E-9) Monoclonal Mouse Santa Cruz Dallas, TX sc-7482 1:1000 Pan-Calcineurin A (PP2B) Polyclonal Rabbit Cell Signaling Danvers, MA 2614 1:1000 PP5 Polyclonal Rabbit Cell Signaling Danvers, MA 2289 1:1000 Gel loading Controls β-actin Monoclonal Mouse Sigma-Aldrich St.Louis, MO A5316 1:10000 β3-tubulin Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 Open in a separate window List of antibodies used 2.3.

Techniques: Phospho-proteomics, Saline, Western Blot

Journal: Neurobiology of aging

Article Title: Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain

doi: 10.1016/j.neurobiolaging.2018.10.027

Figure Lengend Snippet: Hippocampal tau phosphorylation (% Ctl) at the AT8, CP13, PHF-1 phosphoepitopes in 8-10-week-old (A-C) C57BL/6 mice treated with a subcutaneous infusion of midazolam 3 mg/kg/h (n = 5) or 0.9% saline (Ctl; n = 6) and sacrificed 1 week after the start of the 24h infusion. Normothermia was maintained throughout the administration of midazolam or saline. Phosphorylated tau levels were normalized to total tau (D), after controlling for gel loading with β-actin. Relative immunoreactive band intensities are expressed as a percent of Ctl and are displayed for each phosphoepitope and total tau. For each condition, 2 representative immunoblot bands are displayed. Data are expressed as mean ± SD and *, *** denote P < 0.05 and P < 0.001, respectively, vs. Ctl using an unpaired t-test.

Article Snippet: A detailed summary of all of the antibodies used in these studies is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Phospho-Epitope Type Source Manufacturer Location Catalog # WB Dilution Tau Tau46 Total Tau Monoclonal Mouse Cell Signaling Danvers, MA 4019 1:2500 Anti-Human Tau A0024 Total Tau 243-441 Polyclonal Rabbit Dako Cytomation Carpinteria, CA A002401-2 1:10000 TG5 Total Tau 220-240 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT8 pSer202/pThr205 Monoclonal Mouse Thermo Scientific Waltham, MA MN1020 1:10000 CP13 pSer202 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 PHF-1 pSer396/pSer404 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT180 pThr231 Monoclonal Mouse Thermo Scientific Waltham, MA MN1040 1:1000 PS422 pSer422 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-764G 1:1000 PS262 pSer262 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-750G 1:1000 PS199 pSer199 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-734G 1:1000 AT270 pThr181 Monoclonal Mouse Thermo Scientific Waltham, MA MN1050 1:1000 12 E8 pSer262/pSer356 Monoclonal Mouse Pharmaceuticals Francisco, CA N/A 1:1000 Kinases GSK-3β Monoclonal Rabbit Cell Signaling Danvers, MA 9315 1:1000 Phospho-GSK-3 β (Ser9) Ser9 Polyclonal Rabbit Cell Signaling Danvers, MA 9336 1:1000 SAPK/JNK Monoclonal Rabbit Cell Signaling Danvers, MA 9258 1:1000 Phospho-SAPK/JNK Thr183/Tyr185 Monoclonal Rabbit Cell Signaling Danvers, MA 4671 1:1000 p44/42 MAPK Monoclonal Mouse Cell Signaling Danvers, MA 9107 1:2000 Phospho-p44/42 MAPK Thr202/Tyr204 Monoclonal Mouse Cell Signaling Danvers, MA 9106 1:2000 CaMKII Polyclonal Rabbit Cell Signaling Danvers, MA 3362 1:1000 Phospho-CaMKII Thr286 Polyclonal Rabbit Cell Signaling Danvers, MA 3361 1:1000 p35/25 Monoclonal Rabbit Cell Signaling Danvers, MA 2680 1:1000 Phosphatases PP2A-C (catalytic subunit) Monoclonal Mouse Sigma-Aldrich St. Louis, MO A5316 1:10000 demethylated PP2A-C Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 PP1 (E-9) Monoclonal Mouse Santa Cruz Dallas, TX sc-7482 1:1000 Pan-Calcineurin A (PP2B) Polyclonal Rabbit Cell Signaling Danvers, MA 2614 1:1000 PP5 Polyclonal Rabbit Cell Signaling Danvers, MA 2289 1:1000 Gel loading Controls β-actin Monoclonal Mouse Sigma-Aldrich St.Louis, MO A5316 1:10000 β3-tubulin Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 Open in a separate window List of antibodies used 2.3.

Techniques: Phospho-proteomics, Saline, Western Blot

Journal: Neurobiology of aging

Article Title: Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain

doi: 10.1016/j.neurobiolaging.2018.10.027

Figure Lengend Snippet: Hippocampal tau phosphorylation (% Ctl) at the AT8, CP13, and PHF-1 phosphoepitopes (1A-C) in 7-month-old hTau mice treated midazolam 5 mg/kg/h or 0.9% saline (n = 6 per group) and sacrificed 24h after the start of the chronic infusion. Normothermia was maintained throughout the study and rectal temperatures were similar at the time of hippocampal tissue harvest: Ctl 37.3 ± 1.0 °C and Midazolam 36.9 ± 0.3 °C. Phosphorylated tau levels were normalized to total tau (1D), after controlling for gel loading with β-actin. Levels of tau in the sarkosyl-insoluble (SI; 2A), sarkosyl-soluble (Sol; 2B), and heat-stabilized (HS; 2C) and total (TG5) fractions (2D) in cortical homogenates were measured using immunoblotting and total tau (TG5) antibody. Relative immunoreactive band intensities are expressed as a percent of Ctl and are displayed for each phosphoepitope and total tau. For each condition, 2 representative immunoblot bands are displayed. Data are expressed as mean ± SD and * denotes P < 0.05 vs. Ctl using an unpaired t-test.

Article Snippet: A detailed summary of all of the antibodies used in these studies is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Phospho-Epitope Type Source Manufacturer Location Catalog # WB Dilution Tau Tau46 Total Tau Monoclonal Mouse Cell Signaling Danvers, MA 4019 1:2500 Anti-Human Tau A0024 Total Tau 243-441 Polyclonal Rabbit Dako Cytomation Carpinteria, CA A002401-2 1:10000 TG5 Total Tau 220-240 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT8 pSer202/pThr205 Monoclonal Mouse Thermo Scientific Waltham, MA MN1020 1:10000 CP13 pSer202 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 PHF-1 pSer396/pSer404 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT180 pThr231 Monoclonal Mouse Thermo Scientific Waltham, MA MN1040 1:1000 PS422 pSer422 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-764G 1:1000 PS262 pSer262 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-750G 1:1000 PS199 pSer199 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-734G 1:1000 AT270 pThr181 Monoclonal Mouse Thermo Scientific Waltham, MA MN1050 1:1000 12 E8 pSer262/pSer356 Monoclonal Mouse Pharmaceuticals Francisco, CA N/A 1:1000 Kinases GSK-3β Monoclonal Rabbit Cell Signaling Danvers, MA 9315 1:1000 Phospho-GSK-3 β (Ser9) Ser9 Polyclonal Rabbit Cell Signaling Danvers, MA 9336 1:1000 SAPK/JNK Monoclonal Rabbit Cell Signaling Danvers, MA 9258 1:1000 Phospho-SAPK/JNK Thr183/Tyr185 Monoclonal Rabbit Cell Signaling Danvers, MA 4671 1:1000 p44/42 MAPK Monoclonal Mouse Cell Signaling Danvers, MA 9107 1:2000 Phospho-p44/42 MAPK Thr202/Tyr204 Monoclonal Mouse Cell Signaling Danvers, MA 9106 1:2000 CaMKII Polyclonal Rabbit Cell Signaling Danvers, MA 3362 1:1000 Phospho-CaMKII Thr286 Polyclonal Rabbit Cell Signaling Danvers, MA 3361 1:1000 p35/25 Monoclonal Rabbit Cell Signaling Danvers, MA 2680 1:1000 Phosphatases PP2A-C (catalytic subunit) Monoclonal Mouse Sigma-Aldrich St. Louis, MO A5316 1:10000 demethylated PP2A-C Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 PP1 (E-9) Monoclonal Mouse Santa Cruz Dallas, TX sc-7482 1:1000 Pan-Calcineurin A (PP2B) Polyclonal Rabbit Cell Signaling Danvers, MA 2614 1:1000 PP5 Polyclonal Rabbit Cell Signaling Danvers, MA 2289 1:1000 Gel loading Controls β-actin Monoclonal Mouse Sigma-Aldrich St.Louis, MO A5316 1:10000 β3-tubulin Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 Open in a separate window List of antibodies used 2.3.

Techniques: Phospho-proteomics, Saline, Western Blot

Journal: Neurobiology of aging

Article Title: Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain

doi: 10.1016/j.neurobiolaging.2018.10.027

Figure Lengend Snippet: Neocortical brain proteins from 7-month-old hTau mice treated midazolam 5 mg/kg/h or 0.9% saline (n = 6 per group) for 24h were extracted and incubated with taxol-stabilized microtubules. Total tau and μ-tubulin levels from the microtubule free and bound fractions were evaluated by immunoblot analysis. Levels of total tau and μ-tubulin in microtubule free (A,C, respectively) and bound fractions (B,D, respectively) following midazolam treatment were compared to saline (Ctl) after controlling for gel loading with β-actin. Relative immunoreactive band intensities are expressed as a percent of Ctl and, for each condition, 2 representative immunoblot bands are displayed. Data are expressed as mean ± SD and * denotes P < 0.05 vs. Ctl using an unpaired t-test.

Article Snippet: A detailed summary of all of the antibodies used in these studies is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Phospho-Epitope Type Source Manufacturer Location Catalog # WB Dilution Tau Tau46 Total Tau Monoclonal Mouse Cell Signaling Danvers, MA 4019 1:2500 Anti-Human Tau A0024 Total Tau 243-441 Polyclonal Rabbit Dako Cytomation Carpinteria, CA A002401-2 1:10000 TG5 Total Tau 220-240 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT8 pSer202/pThr205 Monoclonal Mouse Thermo Scientific Waltham, MA MN1020 1:10000 CP13 pSer202 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 PHF-1 pSer396/pSer404 Monoclonal Mouse Peter Davies Bronx, NY N/A 1:1000 AT180 pThr231 Monoclonal Mouse Thermo Scientific Waltham, MA MN1040 1:1000 PS422 pSer422 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-764G 1:1000 PS262 pSer262 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-750G 1:1000 PS199 pSer199 Polyclonal Rabbit Life Technologies Carlsbad, CA 44-734G 1:1000 AT270 pThr181 Monoclonal Mouse Thermo Scientific Waltham, MA MN1050 1:1000 12 E8 pSer262/pSer356 Monoclonal Mouse Pharmaceuticals Francisco, CA N/A 1:1000 Kinases GSK-3β Monoclonal Rabbit Cell Signaling Danvers, MA 9315 1:1000 Phospho-GSK-3 β (Ser9) Ser9 Polyclonal Rabbit Cell Signaling Danvers, MA 9336 1:1000 SAPK/JNK Monoclonal Rabbit Cell Signaling Danvers, MA 9258 1:1000 Phospho-SAPK/JNK Thr183/Tyr185 Monoclonal Rabbit Cell Signaling Danvers, MA 4671 1:1000 p44/42 MAPK Monoclonal Mouse Cell Signaling Danvers, MA 9107 1:2000 Phospho-p44/42 MAPK Thr202/Tyr204 Monoclonal Mouse Cell Signaling Danvers, MA 9106 1:2000 CaMKII Polyclonal Rabbit Cell Signaling Danvers, MA 3362 1:1000 Phospho-CaMKII Thr286 Polyclonal Rabbit Cell Signaling Danvers, MA 3361 1:1000 p35/25 Monoclonal Rabbit Cell Signaling Danvers, MA 2680 1:1000 Phosphatases PP2A-C (catalytic subunit) Monoclonal Mouse Sigma-Aldrich St. Louis, MO A5316 1:10000 demethylated PP2A-C Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 PP1 (E-9) Monoclonal Mouse Santa Cruz Dallas, TX sc-7482 1:1000 Pan-Calcineurin A (PP2B) Polyclonal Rabbit Cell Signaling Danvers, MA 2614 1:1000 PP5 Polyclonal Rabbit Cell Signaling Danvers, MA 2289 1:1000 Gel loading Controls β-actin Monoclonal Mouse Sigma-Aldrich St.Louis, MO A5316 1:10000 β3-tubulin Monoclonal Mouse Cell Signaling Danvers, MA 4466 1:1000 Open in a separate window List of antibodies used 2.3.

Techniques: Saline, Incubation, Western Blot

Journal: iScience

Article Title: Non-canonical function of DPP4 promotes cognitive impairment through ERp29-associated mitochondrial calcium overload in diabetes

doi: 10.1016/j.isci.2023.106271

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-ERp29 , Proteintech , Cat#24344-1-AP; RRID: AB_2879503.

Techniques: Virus, Recombinant, SYBR Green Assay, Staining, Immunofluorescence, Blocking Assay, Western Blot, ATP Assay, Isolation, Luciferase, Reporter Gene Assay, Membrane, TUNEL Assay, Apoptosis Assay, Bicinchoninic Acid Protein Assay, Activity Assay, Plasmid Preparation, Software

Journal: Molecular Pharmacology

Article Title: Transient receptor potential ankyrin 1 promotes the expression of interferon-stimulated antiviral genes in human A549 lung epithelial cells

doi: 10.1016/j.molpha.2025.100098

Figure Lengend Snippet: TRPA1 antagonists inhibit IFN-beta-induced STAT1 tyrosine phosphorylation at the position 701. A549 cells were cultured for 15 minutes with 10 ng/mL of IFN beta (A, C) or IFN alfa-2a (B, D), with or without the TRPA1 antagonists HC-030031 (HC; 100 μ M) or A-967079 (A96; 100 μ M). Thereafter, total protein was extracted and western blot of tyrosine phosphorylated STAT1 (at the position 701; pSTAT1-Y701) and total STAT1 was performed. In panels A and B, representative blots (with 2 biological replicates of each treatment) are shown. In panels C and D, densitometric data of pSTAT1-Y701 normalized to the total STAT1 signal of the respective sample are presented as individual values and mean ± SD, n = 6. Statistical analysis was performed using one-way ANOVA with Holm- Šídák post-test against the IFN-treated condition (second bar from the left). ∗ and ∗∗∗∗ indicate P < .05 and P < .0001, respectively.

Article Snippet: The following primary antibodies diluted 1:1000 in 5% BSA were used: OAS2 (E2G4K) Rabbit mAb #24344, MX1 (D3W7I) Rabbit mAb #37849, Rig-I (D33H10) Rabbit mAb #4200, Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb #7649, and Stat1 antibody #9172 (all from Cell Signaling Technology, CST).

Techniques: Phospho-proteomics, Cell Culture, Western Blot

Journal: Molecular Pharmacology

Article Title: Transient receptor potential ankyrin 1 promotes the expression of interferon-stimulated antiviral genes in human A549 lung epithelial cells

doi: 10.1016/j.molpha.2025.100098

Figure Lengend Snippet: The effect of TRPA1 antagonists on the mRNA expression of the IFN-stimulated antiviral genes MX1, OAS2, and DDX58 . A549 cells were cultured with IFN beta or IFN alfa-2a (both 10 ng/mL) in the presence or absence of the TRPA1 antagonists HC-030031 or A-967079 at indicated concentrations for 24 hours. Thereafter, total RNA was extracted, and RT-qPCR of MX1 , OAS2, and DDX58 was carried out against RPL30 as an endogenous control gene. Individual values and mean ± SD are presented, n = 5–6. Control was set as 1, and the other values are given in relation to that value. Statistical analysis was performed using one-way ANOVA with Holm- Šídák post-test, and comparisons were carried out against the IFN-treated condition (second bar from the left). ∗, ∗∗, ∗∗∗, and ∗∗∗∗ denote P < .05, <.01, <.001, or <.0001, respectively.

Article Snippet: The following primary antibodies diluted 1:1000 in 5% BSA were used: OAS2 (E2G4K) Rabbit mAb #24344, MX1 (D3W7I) Rabbit mAb #37849, Rig-I (D33H10) Rabbit mAb #4200, Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb #7649, and Stat1 antibody #9172 (all from Cell Signaling Technology, CST).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Control

Journal: Molecular Pharmacology

Article Title: Transient receptor potential ankyrin 1 promotes the expression of interferon-stimulated antiviral genes in human A549 lung epithelial cells

doi: 10.1016/j.molpha.2025.100098

Figure Lengend Snippet: The effect of the TRPA1 antagonists on the protein expression of the IFN-stimulated antiviral genes MX1, OAS2 . and DDX58 . A549 cells were cultured with IFN beta (10 ng/mL; A, C) or IFN alfa-2a (10 ng/mL; B, D) for 24 hours with or without the TRPA1 antagonists HC-030031 (HC; 100 μ M) or A-967079 (A96; 100 μ M), total protein was extracted, and indicated proteins were detected by western blotting. In panels A and B, representative blots of the indicated proteins (with 2 biological replicates of each treatment) are shown following IFN beta (A) and IFN alfa-2a (B) treatment, respectively. Actin was detected as a loading control. Densitometric analysis is shown in panels C and D. Individual values and mean ± SD are presented, n = 5–6. IFN-treated conditions (second bar from the left) were set as 1, and the other values are given in relation to and statistically tested against that value. Statistical analysis was performed using one-way ANOVA with Holm- Šídák post-test. ∗, ∗∗, ∗∗∗, and ∗∗∗∗ denote P < .05, <.01, <.001, or <.0001, respectively.

Article Snippet: The following primary antibodies diluted 1:1000 in 5% BSA were used: OAS2 (E2G4K) Rabbit mAb #24344, MX1 (D3W7I) Rabbit mAb #37849, Rig-I (D33H10) Rabbit mAb #4200, Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb #7649, and Stat1 antibody #9172 (all from Cell Signaling Technology, CST).

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Molecular Pharmacology

Article Title: Transient receptor potential ankyrin 1 promotes the expression of interferon-stimulated antiviral genes in human A549 lung epithelial cells

doi: 10.1016/j.molpha.2025.100098

Figure Lengend Snippet: MX1 induction by IFN beta is inhibited by genetic TRPA1 knockdown. (A, B) A549 cells were transfected with TRPA1 siRNA, nontargeting (negative control; Neg) siRNA or were not transfected. After 72 hours, the cells were cultured with IFN beta (10 ng/mL) with or without the TRPA1 antagonist A-967079 (100 μ M) for 24 hours. Thereafter, total RNA was extracted, and RT-qPCR was performed. RPL30 was used as an endogenous control gene. In panel A is shown the level of MX1 expression relative to the respective controls of each transfection condition which were set as 1. In panel B, TRPA1 knockdown by siRNA relative to Neg siRNA (which was set as 1) is shown. N = 5–6 (A) and n = 6 (B). In panel C, a summary of Mx1 expression data from ex vivo experiments is presented. Lung tissue cultures from WT ( n = 10) and TRPA1-deficient (KO; n = 7) mice were exposed to IFN beta (10 ng/mL) for 24 hours, tissue pieces were homogenized, total RNA was extracted, and Mx1 expression was measured by RT-qPCR. Gapdh was used as an endogenous control gene. The control of each mouse was set as 1, and the treated condition of the same mouse was given in relation to that value. In all figures, individual values and mean ± SD are presented. Statistical analysis was carried out using one-way ANOVA with Holm- Šídák post-test (A) or Student’s t test (B). In panel C, two-way repeated measures ANOVA (mixed effects model) with Holm- Šídák post-test was used and the interaction between the genotype and the treatment was statistically significant ( P < .05). ∗∗ and ∗∗∗∗ denote <.01 and .0001, respectively.

Article Snippet: The following primary antibodies diluted 1:1000 in 5% BSA were used: OAS2 (E2G4K) Rabbit mAb #24344, MX1 (D3W7I) Rabbit mAb #37849, Rig-I (D33H10) Rabbit mAb #4200, Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb #7649, and Stat1 antibody #9172 (all from Cell Signaling Technology, CST).

Techniques: Knockdown, Transfection, Negative Control, Cell Culture, Quantitative RT-PCR, Control, Expressing, Ex Vivo