Journal: iScience

Article Title: Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes

doi: 10.1016/j.isci.2023.107680

Figure Lengend Snippet: Involvement of intracellular Ca 2+ , CaM, calcineurin and PP1 in suppression of I LCa by Ex-4 (A and B) Representative recordings from an RGC at −10 mV, showing that during internal infusion of Ca 2+ -free solution (containing 10 mM BAPTA; control), Ex-4 failed to suppress the I LCa , and summary data are shown in (B) (n = 6). (C and E) Current traces of two RGCs, showing that during internal dialysis of 5 mg/mL heparin (C), but not ryanodine (50 μM, E), the Ex-4-induced suppression of I LCa was seen. (D and F) Bar charts summarizing the effects of Ex-4 on the I LCa amplitudes in the presence of heparin (n = 7) (D) or ryanodine (n = 7) (F). (G, I, and K) Representative recordings obtained from three different RGCs showing that Ex-4 no longer reduced the I LCa amplitudes during the internal infusion of 100 μM W-7 (G), 50 μM FK-506 (I) and 1 μM OA (K) to block CaM, calcineurin and PP1, respectively. (H, J, and L) Bar charts summarizing the results regarding the effects of W-7 (n = 7) (H), FK-506 (n = 8) (J) or OA (n = 8) (L). (M and N) Current traces of an RGC, showing that perfusion of C2 Ceramide (500 nM) suppressed the I LCa (M), and summary data are shown in (N) (n = 6). Data are presented as mean ± SD, n.s., p > 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 by paired t test. (O) Representative immunoblots showed changes in the expression of L-VGCC subunits Cav1.2 and p -Cav1.2 in membrane components in retinas from the control, DM rats treated with or without Ex-4 eye drops. Na + -K + -ATPase served as loading control. (P and Q) Densitometric analysis revealing a significant increase in Cav1.2 (P) and p -Cav1.2 (Q) protein levels in DM retinas. Topical administration of Ex-4 significantly decreased Cav1.2 and p -Cav1.2 levels in DM retinas. (R) Quantitative analysis of p -Cav1.2 relative to Cav1.2 total protein is shown in the bar graph. The level of p -Cav1.2/Cav1.2 did not change in DM retinas, but significantly decreased in the DM + Ex-4 group. n = 3 for each group. Data are presented as mean ± SD. n.s., p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 by post hoc Tukey’s multiple comparisons test after one-way ANOVA. See also Figure S3 .

Article Snippet: Equivalent amounts of freshly extracted samples were loaded (for the detection of p-Cav1.2, the loading sample volume was 3 times that of the detection of Cav1.2, due to the low content of p-Cav1.2), subjected to 8% SDS-PAGE, and then transferred onto PVDF membranes (Immobilon-P, Millipore Co. Bedford, MA, USA).

Techniques: Blocking Assay, Western Blot, Expressing, Membrane, Eye Drops

Journal: iScience

Article Title: Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes

doi: 10.1016/j.isci.2023.107680

Figure Lengend Snippet:

Article Snippet: Equivalent amounts of freshly extracted samples were loaded (for the detection of p-Cav1.2, the loading sample volume was 3 times that of the detection of Cav1.2, due to the low content of p-Cav1.2), subjected to 8% SDS-PAGE, and then transferred onto PVDF membranes (Immobilon-P, Millipore Co. Bedford, MA, USA).

Techniques: Recombinant, Protease Inhibitor, Software

Journal: iScience

Article Title: Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes

doi: 10.1016/j.isci.2023.107680

Figure Lengend Snippet: Involvement of intracellular Ca 2+ , CaM, calcineurin and PP1 in suppression of I LCa by Ex-4 (A and B) Representative recordings from an RGC at −10 mV, showing that during internal infusion of Ca 2+ -free solution (containing 10 mM BAPTA; control), Ex-4 failed to suppress the I LCa , and summary data are shown in (B) (n = 6). (C and E) Current traces of two RGCs, showing that during internal dialysis of 5 mg/mL heparin (C), but not ryanodine (50 μM, E), the Ex-4-induced suppression of I LCa was seen. (D and F) Bar charts summarizing the effects of Ex-4 on the I LCa amplitudes in the presence of heparin (n = 7) (D) or ryanodine (n = 7) (F). (G, I, and K) Representative recordings obtained from three different RGCs showing that Ex-4 no longer reduced the I LCa amplitudes during the internal infusion of 100 μM W-7 (G), 50 μM FK-506 (I) and 1 μM OA (K) to block CaM, calcineurin and PP1, respectively. (H, J, and L) Bar charts summarizing the results regarding the effects of W-7 (n = 7) (H), FK-506 (n = 8) (J) or OA (n = 8) (L). (M and N) Current traces of an RGC, showing that perfusion of C2 Ceramide (500 nM) suppressed the I LCa (M), and summary data are shown in (N) (n = 6). Data are presented as mean ± SD, n.s., p > 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 by paired t test. (O) Representative immunoblots showed changes in the expression of L-VGCC subunits Cav1.2 and p -Cav1.2 in membrane components in retinas from the control, DM rats treated with or without Ex-4 eye drops. Na + -K + -ATPase served as loading control. (P and Q) Densitometric analysis revealing a significant increase in Cav1.2 (P) and p -Cav1.2 (Q) protein levels in DM retinas. Topical administration of Ex-4 significantly decreased Cav1.2 and p -Cav1.2 levels in DM retinas. (R) Quantitative analysis of p -Cav1.2 relative to Cav1.2 total protein is shown in the bar graph. The level of p -Cav1.2/Cav1.2 did not change in DM retinas, but significantly decreased in the DM + Ex-4 group. n = 3 for each group. Data are presented as mean ± SD. n.s., p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 by post hoc Tukey’s multiple comparisons test after one-way ANOVA. See also Figure S3 .

Article Snippet: Equivalent amounts of freshly extracted samples were loaded (for the detection of p-Cav1.2, the loading sample volume was 3 times that of the detection of Cav1.2, due to the low content of p-Cav1.2), subjected to 8% SDS-PAGE, and then transferred onto PVDF membranes (Immobilon-P, Millipore Co. Bedford, MA, USA).

Techniques: Blocking Assay, Western Blot, Expressing, Membrane, Eye Drops

Journal: iScience

Article Title: Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes

doi: 10.1016/j.isci.2023.107680

Figure Lengend Snippet:

Article Snippet: Equivalent amounts of freshly extracted samples were loaded (for the detection of p-Cav1.2, the loading sample volume was 3 times that of the detection of Cav1.2, due to the low content of p-Cav1.2), subjected to 8% SDS-PAGE, and then transferred onto PVDF membranes (Immobilon-P, Millipore Co. Bedford, MA, USA).

Techniques: Recombinant, Protease Inhibitor, Software

Journal: The Journal of Neuroscience

Article Title: Mechanism of Manganese Dysregulation of Dopamine Neuronal Activity

doi: 10.1523/JNEUROSCI.2830-19.2020

Figure Lengend Snippet: Antibodies for immunofluorescence staining and Western blot

Article Snippet: Cav1.2 antibody , Mouse IgG2bκ , 1:1000 , Millipore, MAB13170.

Techniques: Immunofluorescence, Staining, Western Blot

Journal: The Journal of Neuroscience

Article Title: Mechanism of Manganese Dysregulation of Dopamine Neuronal Activity

doi: 10.1523/JNEUROSCI.2830-19.2020

Figure Lengend Snippet: Superposition of msCav1.1 Ca binding site structure with the msCav1.2 model. The Ca2+ ion (green) as located in the msCav1.1 structure (tan color backbone) (PDB ID 5GJW). The homology model structure for Cav1.2 α chain is shown in the blue color backbone.

Article Snippet: Cav1.2 antibody , Mouse IgG2bκ , 1:1000 , Millipore, MAB13170.

Techniques: Binding Assay

Journal: The Journal of Neuroscience

Article Title: Mechanism of Manganese Dysregulation of Dopamine Neuronal Activity

doi: 10.1523/JNEUROSCI.2830-19.2020

Figure Lengend Snippet: 54Mn uptake is greater in Cav1.2-overexpressing HEK293 cells. A, Representative Western blot analysis shows the expression of Cav1.2 and Cav1.3 in cells transiently transfected to overexpress Cav1.2 and Cav1.3. B, Greater 54Mn uptake is shown in Cav1.2-overexpressing cells at the indicated time points. C, Nifepidine inhibits 54Mn uptake at 15 min in Cav1.2-overexpressing cells. *p < 0.05 (one-way ANOVA followed by Tukey's test). n = 3 per group.

Article Snippet: Cav1.2 antibody , Mouse IgG2bκ , 1:1000 , Millipore, MAB13170.

Techniques: Western Blot, Expressing, Transfection

Journal: The Journal of Neuroscience

Article Title: Mechanism of Manganese Dysregulation of Dopamine Neuronal Activity

doi: 10.1523/JNEUROSCI.2830-19.2020

Figure Lengend Snippet: Antibodies for immunofluorescence staining and Western blot

Article Snippet: The catalog number of the reagents and drugs used for electrophysiology and microscopy experiments are listed in . table ft1 table-wrap mode="anchored" t5 caption a7 Chemical Concentration (m m ) Manufacturer, catalog # NaCl 116 Millipore Sigma, S7653 KCl 5.4 Millipore Sigma, P9541 NaHCO 3 26 Millipore Sigma, S5761 D-(+)-Glucose 25 Millipore Sigma, G8270 NaH 2 PO 4 2 Millipore Sigma, 71505 MgSO 4 1 Millipore Sigma, M7506 Cysteine 1.3 Millipore Sigma, C6645 EDTA 0.5 Millipore Sigma, E9884 Kynurenate 0.5 Millipore Sigma, K3375 Papain 20 units/ml Worthington, {"type":"entrez-nucleotide","attrs":{"text":"LS003126","term_id":"1321651598","term_text":"LS003126"}} LS003126 Penicillin/streptomycin 7.70% Millipore Sigma, P4333 L-glutamine 0.90% Millipore Sigma, 59202C Poly-L-lysine 100 μg/ml Millipore Sigma, P1399 Laminin 5 μg/ml Millipore Sigma, L2020 Insulin 0.025 g/ml Millipore Sigma, I2643 Apotransferrin 50 ml/ml Millipore Sigma, T0178 GDNF 1 ng/ml Millipore Sigma, SRP3200 Open in a separate window Chemical reagents and drugs for primary neuronal culture table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host species Dilution Manufacturer, catalog # Cav1.2 antibody Mouse IgG2bκ 1:1000 Millipore, MAB13170 Anti-Ca(v)1.3 alpha1 subunit antibody Rabbit 1:500 Millipore, ABN11 Open in a separate window Antibodies for immunofluorescence staining and Western blot table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Chemical Concentration (m m ) Manufacturer, catalog # NaCl 126 Millipore Sigma, S7653 KCl 2.5 Millipore Sigma, P9541 CaC l2 2 Millipore Sigma, 223506 NaHCO 3 26 Millipore Sigma, S5761 NaH 2 PO 4 1.25 Millipore Sigma, 71505 MgSO 4 2 Millipore Sigma, M7506 Dextrose 10 Millipore Sigma, D9434 Potassium gluconate 120 Millipore Sigma, P1847 MgCl 2 2 Millipore Sigma, M8266 HEPES 10 Millipore Sigma, H3375 EGTA 0.1 Millipore Sigma, E3889 ATPNa 2 2 Millipore Sigma, A2383 GTPNa 0.25 Millipore Sigma, G8877 CsMeSO 3 110 Millipore Sigma, C1426 Cesium chloride 1 Millipore Sigma, 289329 Tetraethylammonium 10 Millipore Sigma, T2265 Tetrodotoxin 0.0005 Cayman, 14964 4-Aminopyridine 1 Millipore Sigma, 275875 Manganese chloride 0.1 Thermo Fisher Scientific, M87-100 Paxilline 0.01 Tocris Bioscience, 2006 Nifedipine 15 mg/kg Millipore Sigma, N7634 BAPTA-AM 10 Millipore Sigma, A1076 NMDA 0.1 Millipore Sigma, M3262 Cadmium chloride 0.1 Millipore Sigma, 202908 fura-2 AM 0.005 Thermo Fisher Scientific, F1201 Caffeine 20 Millipore Sigma, C0750 Open in a separate window Chemical reagents and drugs for electrophysiological and TIRF recordings

Techniques: Immunofluorescence, Staining, Western Blot

Journal: The Journal of Neuroscience

Article Title: Mechanism of Manganese Dysregulation of Dopamine Neuronal Activity

doi: 10.1523/JNEUROSCI.2830-19.2020

Figure Lengend Snippet: Superposition of msCav1.1 Ca binding site structure with the msCav1.2 model. The Ca2+ ion (green) as located in the msCav1.1 structure (tan color backbone) (PDB ID 5GJW). The homology model structure for Cav1.2 α chain is shown in the blue color backbone.

Article Snippet: The catalog number of the reagents and drugs used for electrophysiology and microscopy experiments are listed in . table ft1 table-wrap mode="anchored" t5 caption a7 Chemical Concentration (m m ) Manufacturer, catalog # NaCl 116 Millipore Sigma, S7653 KCl 5.4 Millipore Sigma, P9541 NaHCO 3 26 Millipore Sigma, S5761 D-(+)-Glucose 25 Millipore Sigma, G8270 NaH 2 PO 4 2 Millipore Sigma, 71505 MgSO 4 1 Millipore Sigma, M7506 Cysteine 1.3 Millipore Sigma, C6645 EDTA 0.5 Millipore Sigma, E9884 Kynurenate 0.5 Millipore Sigma, K3375 Papain 20 units/ml Worthington, {"type":"entrez-nucleotide","attrs":{"text":"LS003126","term_id":"1321651598","term_text":"LS003126"}} LS003126 Penicillin/streptomycin 7.70% Millipore Sigma, P4333 L-glutamine 0.90% Millipore Sigma, 59202C Poly-L-lysine 100 μg/ml Millipore Sigma, P1399 Laminin 5 μg/ml Millipore Sigma, L2020 Insulin 0.025 g/ml Millipore Sigma, I2643 Apotransferrin 50 ml/ml Millipore Sigma, T0178 GDNF 1 ng/ml Millipore Sigma, SRP3200 Open in a separate window Chemical reagents and drugs for primary neuronal culture table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host species Dilution Manufacturer, catalog # Cav1.2 antibody Mouse IgG2bκ 1:1000 Millipore, MAB13170 Anti-Ca(v)1.3 alpha1 subunit antibody Rabbit 1:500 Millipore, ABN11 Open in a separate window Antibodies for immunofluorescence staining and Western blot table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Chemical Concentration (m m ) Manufacturer, catalog # NaCl 126 Millipore Sigma, S7653 KCl 2.5 Millipore Sigma, P9541 CaC l2 2 Millipore Sigma, 223506 NaHCO 3 26 Millipore Sigma, S5761 NaH 2 PO 4 1.25 Millipore Sigma, 71505 MgSO 4 2 Millipore Sigma, M7506 Dextrose 10 Millipore Sigma, D9434 Potassium gluconate 120 Millipore Sigma, P1847 MgCl 2 2 Millipore Sigma, M8266 HEPES 10 Millipore Sigma, H3375 EGTA 0.1 Millipore Sigma, E3889 ATPNa 2 2 Millipore Sigma, A2383 GTPNa 0.25 Millipore Sigma, G8877 CsMeSO 3 110 Millipore Sigma, C1426 Cesium chloride 1 Millipore Sigma, 289329 Tetraethylammonium 10 Millipore Sigma, T2265 Tetrodotoxin 0.0005 Cayman, 14964 4-Aminopyridine 1 Millipore Sigma, 275875 Manganese chloride 0.1 Thermo Fisher Scientific, M87-100 Paxilline 0.01 Tocris Bioscience, 2006 Nifedipine 15 mg/kg Millipore Sigma, N7634 BAPTA-AM 10 Millipore Sigma, A1076 NMDA 0.1 Millipore Sigma, M3262 Cadmium chloride 0.1 Millipore Sigma, 202908 fura-2 AM 0.005 Thermo Fisher Scientific, F1201 Caffeine 20 Millipore Sigma, C0750 Open in a separate window Chemical reagents and drugs for electrophysiological and TIRF recordings

Techniques: Binding Assay

Journal: The Journal of Neuroscience

Article Title: Mechanism of Manganese Dysregulation of Dopamine Neuronal Activity

doi: 10.1523/JNEUROSCI.2830-19.2020

Figure Lengend Snippet: 54Mn uptake is greater in Cav1.2-overexpressing HEK293 cells. A, Representative Western blot analysis shows the expression of Cav1.2 and Cav1.3 in cells transiently transfected to overexpress Cav1.2 and Cav1.3. B, Greater 54Mn uptake is shown in Cav1.2-overexpressing cells at the indicated time points. C, Nifepidine inhibits 54Mn uptake at 15 min in Cav1.2-overexpressing cells. *p < 0.05 (one-way ANOVA followed by Tukey's test). n = 3 per group.

Article Snippet: The catalog number of the reagents and drugs used for electrophysiology and microscopy experiments are listed in . table ft1 table-wrap mode="anchored" t5 caption a7 Chemical Concentration (m m ) Manufacturer, catalog # NaCl 116 Millipore Sigma, S7653 KCl 5.4 Millipore Sigma, P9541 NaHCO 3 26 Millipore Sigma, S5761 D-(+)-Glucose 25 Millipore Sigma, G8270 NaH 2 PO 4 2 Millipore Sigma, 71505 MgSO 4 1 Millipore Sigma, M7506 Cysteine 1.3 Millipore Sigma, C6645 EDTA 0.5 Millipore Sigma, E9884 Kynurenate 0.5 Millipore Sigma, K3375 Papain 20 units/ml Worthington, {"type":"entrez-nucleotide","attrs":{"text":"LS003126","term_id":"1321651598","term_text":"LS003126"}} LS003126 Penicillin/streptomycin 7.70% Millipore Sigma, P4333 L-glutamine 0.90% Millipore Sigma, 59202C Poly-L-lysine 100 μg/ml Millipore Sigma, P1399 Laminin 5 μg/ml Millipore Sigma, L2020 Insulin 0.025 g/ml Millipore Sigma, I2643 Apotransferrin 50 ml/ml Millipore Sigma, T0178 GDNF 1 ng/ml Millipore Sigma, SRP3200 Open in a separate window Chemical reagents and drugs for primary neuronal culture table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host species Dilution Manufacturer, catalog # Cav1.2 antibody Mouse IgG2bκ 1:1000 Millipore, MAB13170 Anti-Ca(v)1.3 alpha1 subunit antibody Rabbit 1:500 Millipore, ABN11 Open in a separate window Antibodies for immunofluorescence staining and Western blot table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Chemical Concentration (m m ) Manufacturer, catalog # NaCl 126 Millipore Sigma, S7653 KCl 2.5 Millipore Sigma, P9541 CaC l2 2 Millipore Sigma, 223506 NaHCO 3 26 Millipore Sigma, S5761 NaH 2 PO 4 1.25 Millipore Sigma, 71505 MgSO 4 2 Millipore Sigma, M7506 Dextrose 10 Millipore Sigma, D9434 Potassium gluconate 120 Millipore Sigma, P1847 MgCl 2 2 Millipore Sigma, M8266 HEPES 10 Millipore Sigma, H3375 EGTA 0.1 Millipore Sigma, E3889 ATPNa 2 2 Millipore Sigma, A2383 GTPNa 0.25 Millipore Sigma, G8877 CsMeSO 3 110 Millipore Sigma, C1426 Cesium chloride 1 Millipore Sigma, 289329 Tetraethylammonium 10 Millipore Sigma, T2265 Tetrodotoxin 0.0005 Cayman, 14964 4-Aminopyridine 1 Millipore Sigma, 275875 Manganese chloride 0.1 Thermo Fisher Scientific, M87-100 Paxilline 0.01 Tocris Bioscience, 2006 Nifedipine 15 mg/kg Millipore Sigma, N7634 BAPTA-AM 10 Millipore Sigma, A1076 NMDA 0.1 Millipore Sigma, M3262 Cadmium chloride 0.1 Millipore Sigma, 202908 fura-2 AM 0.005 Thermo Fisher Scientific, F1201 Caffeine 20 Millipore Sigma, C0750 Open in a separate window Chemical reagents and drugs for electrophysiological and TIRF recordings

Techniques: Western Blot, Expressing, Transfection

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Hydrogen sulfide inhibits Ca 2+ -induced mitochondrial permeability transition pore opening in type-1 diabetes

doi: 10.1152/ajpendo.00251.2018

Figure Lengend Snippet: Expression of Cav1.2 protein in kidney tissue and in mouse glomerular endothelial cells (MGECs). A: Cav1.2 expression in wild-type (WT) and Akita kidney cortical tissue with or without GYY4137 (GYY). B: MGECs were incubated without or with MK-801, a selective and noncompetitive antagonist of N-methyl-d-aspartate receptor (NMDA-R) in high-glucose (HG) and normal glucose (NG) conditions, and GYY was administered in the groups as indicated in the figure. Cell lysates were analyzed by immunoblotting against Cav1.2 antibody. Bar diagrams represent mean ± SE, n = 5 or 6; *,† <0.05 and ϕP > 0.05.

Article Snippet: The protein lysates that were subjected to Western blotting and immunofluorescence studies with specific antibodies were purchased as follows: Anti-Cav1.2 (MAB-13170) from Millipore (Burlington, MA); Cyclophilin D (PA3-022), NMDA-R1 (PA3-102), and TIM-1 (KIM-1) PA5-20244 from Thermo Fisher Scientific (Waltham, MA); β-actin (SC-47778) and horseradish peroxidase-linked secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Incubation, Western Blot