Journal: American Journal of Physiology - Renal Physiology

Article Title: Dual gain and loss of cullin 3 function mediates familial hyperkalemic hypertension

doi: 10.1152/ajprenal.00602.2017

Figure Lengend Snippet: Antibodies used for Western blot

Article Snippet: Antibodies used are described in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Figure Target Antibody Name Source, cat. no. 1° Dilution 2° Dilution , JAB1 JAB1 FL-334 Santa Cruz, SC-9074 1:1,000, 1 h at RT 1:5,000 , C and D JAB1 JAB1 6C3.38 Thermo Fisher Scientific 1:2,000, 1 h at RT 1:5,000 GST GST B-14 Santa Cruz, SC-138 1:1,000, 1 h at RT 1:2,500 , , , , , , , Myc c-Myc Sigma-Aldrich, M5546 1:5,000, 1 h at RT 1:10,000 , , NEDD8 NEDD8 19E3 Cell Signaling, 2754 1:1,000, o/n at 4°C 1:2,500 , Cul3 Cul3 Cell Signaling, 2759 1:1,000, o/n at 4°C 1:2,500 , β-actin β-actin Abcam, Ab8227 1:5,000, 1 h at RT 1:2,500 , , , , , , FLAG FLAG M2 Sigma-Aldrich, F3165 1:10,000, 1 h at RT 1:10,000 HA HA.11 Covance, MMS-101P 1:1,000, 1 h at RT 1:10,000 , GAPDH GAPDH Santa Cruz, SC-20357 1:1,000, 1 h at RT 1:2,500 Keap1 Keap1 Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab139729","term_id":"62160310","term_text":"AB139729"}} Ab139729 1:1,000, o/n at 4°C 1:2,500 Nrf2 Nrf2 H-300 Santa Cruz, SC-13032 1:1,000, o/n at 4°C 1:2,500 Cyclin E Cyclin E HE12 Santa Cruz, SC-247 1:1,000, o/n at 4°C 1:2,500 Open in a separate window Cul3, cullin 3; GST, glutathione S-transferase; HA, hemagglutinin; JAB1, jun activation domain-binding protein-1; Keap1, kelch-like ECH-associated protein 1; NEDD8, neuronal precursor cell expressed developmentally downregulated protein 8; Nrf2, nuclear factor erythroid 2-related factor 2; o/n, overnight; RT, room temperature.

Techniques: Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Dual gain and loss of cullin 3 function mediates familial hyperkalemic hypertension

doi: 10.1152/ajprenal.00602.2017

Figure Lengend Snippet: CSN binds to Cul3 at the α/β1 domain. A: Diagram of Cul3 domain structure and schematic of the Cul3 constructs. B: Coimmunoprecipitation was performed with HEK293 cells transfected with myc-JAB1 and FLAG-tagged WT Cul3 or Cul3Δ403–459 and analyzed by immunoblot. Cul3Δ403–459 exhibited a decreased interaction with JAB1 compared with WT Cul3. C: Effects of Cul3Δ403–459 on JAB1 binding was determined by coimmunoprecipitation of HEK293 cells with N-terminal domain Cul3 constructs using anti-FLAG and analyzed by immunoblot. Coimmunoprecipitation of N-terminal domain Cul3 constructs with (1–459) and without (1–402) the 4HB domain showed no binding to JAB1. D: Segments of the Cul3 protein were generated with a GST tag and cotransfected with myc-tagged JAB1 in HEK293 cells. Coimmunoprecipitation was performed using glutathione sepharose beads. Immunoblotting for JAB1 showed binding to 4HB:α/β1 and α/β1 Cul3 constructs but not to 4HB, WH-A:α/β:WH-B, or R1:R2:R3 Cul3 constructs. E: Coimmunoprecipitation was performed in HEK293 cells with myc-JAB1 and FLAG-tagged WT Cul3 or Cul3Δ461–586 constructs. Cul3Δ461–586 demonstrated less binding to JAB1 protein compared with WT Cul3. Immunoblotting for NEDD8 showed enhanced neddylation of the Cul3Δ461–586 construct compared with WT Cul3. *Nonspecific band. 4HB, 4-helix bundle; Cul3, cullin 3; CSN, COP9 signalosome; GST, glutathione S-transferase; HEK, human embryonic kidney; IP, immunoprecipitation; JAB1, jun activation domain-binding protein-1; NEDD8, neuronal precursor cell expressed developmentally downregulated protein 8; WT, wild type.

Article Snippet: Antibodies used are described in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Figure Target Antibody Name Source, cat. no. 1° Dilution 2° Dilution , JAB1 JAB1 FL-334 Santa Cruz, SC-9074 1:1,000, 1 h at RT 1:5,000 , C and D JAB1 JAB1 6C3.38 Thermo Fisher Scientific 1:2,000, 1 h at RT 1:5,000 GST GST B-14 Santa Cruz, SC-138 1:1,000, 1 h at RT 1:2,500 , , , , , , , Myc c-Myc Sigma-Aldrich, M5546 1:5,000, 1 h at RT 1:10,000 , , NEDD8 NEDD8 19E3 Cell Signaling, 2754 1:1,000, o/n at 4°C 1:2,500 , Cul3 Cul3 Cell Signaling, 2759 1:1,000, o/n at 4°C 1:2,500 , β-actin β-actin Abcam, Ab8227 1:5,000, 1 h at RT 1:2,500 , , , , , , FLAG FLAG M2 Sigma-Aldrich, F3165 1:10,000, 1 h at RT 1:10,000 HA HA.11 Covance, MMS-101P 1:1,000, 1 h at RT 1:10,000 , GAPDH GAPDH Santa Cruz, SC-20357 1:1,000, 1 h at RT 1:2,500 Keap1 Keap1 Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab139729","term_id":"62160310","term_text":"AB139729"}} Ab139729 1:1,000, o/n at 4°C 1:2,500 Nrf2 Nrf2 H-300 Santa Cruz, SC-13032 1:1,000, o/n at 4°C 1:2,500 Cyclin E Cyclin E HE12 Santa Cruz, SC-247 1:1,000, o/n at 4°C 1:2,500 Open in a separate window Cul3, cullin 3; GST, glutathione S-transferase; HA, hemagglutinin; JAB1, jun activation domain-binding protein-1; Keap1, kelch-like ECH-associated protein 1; NEDD8, neuronal precursor cell expressed developmentally downregulated protein 8; Nrf2, nuclear factor erythroid 2-related factor 2; o/n, overnight; RT, room temperature.

Techniques: Construct, Transfection, Western Blot, Binding Assay, Generated, Immunoprecipitation, Activation Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Dual gain and loss of cullin 3 function mediates familial hyperkalemic hypertension

doi: 10.1152/ajprenal.00602.2017

Figure Lengend Snippet: Effects of JAB1 inhibition on Cul3 neddylation and substrate protein abundance. Myc-tagged KLHL3 or WNK4 was cotransfected into HEK293 cells with either JAB1 siRNA or control siRNA. The proteins were examined by immunoblot in cells with endogenous WT Cul3. JAB1 siRNA decreased JAB1, KLHL3, and WNK4 abundance and increased NEDD8 abundance and the neddylated form of Cul3 (top band). β-actin was used as a loading control. Con, control; Cul3, cullin 3; HEK, human embryonic kidney; JAB1, jun activation domain-binding protein-1; KLHL3, kelch-like 3; NEDD8, neuronal precursor cell expressed developmentally downregulated protein 8; WNK, with-no-lysine kinase; WT, wild type.

Article Snippet: Antibodies used are described in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Figure Target Antibody Name Source, cat. no. 1° Dilution 2° Dilution , JAB1 JAB1 FL-334 Santa Cruz, SC-9074 1:1,000, 1 h at RT 1:5,000 , C and D JAB1 JAB1 6C3.38 Thermo Fisher Scientific 1:2,000, 1 h at RT 1:5,000 GST GST B-14 Santa Cruz, SC-138 1:1,000, 1 h at RT 1:2,500 , , , , , , , Myc c-Myc Sigma-Aldrich, M5546 1:5,000, 1 h at RT 1:10,000 , , NEDD8 NEDD8 19E3 Cell Signaling, 2754 1:1,000, o/n at 4°C 1:2,500 , Cul3 Cul3 Cell Signaling, 2759 1:1,000, o/n at 4°C 1:2,500 , β-actin β-actin Abcam, Ab8227 1:5,000, 1 h at RT 1:2,500 , , , , , , FLAG FLAG M2 Sigma-Aldrich, F3165 1:10,000, 1 h at RT 1:10,000 HA HA.11 Covance, MMS-101P 1:1,000, 1 h at RT 1:10,000 , GAPDH GAPDH Santa Cruz, SC-20357 1:1,000, 1 h at RT 1:2,500 Keap1 Keap1 Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab139729","term_id":"62160310","term_text":"AB139729"}} Ab139729 1:1,000, o/n at 4°C 1:2,500 Nrf2 Nrf2 H-300 Santa Cruz, SC-13032 1:1,000, o/n at 4°C 1:2,500 Cyclin E Cyclin E HE12 Santa Cruz, SC-247 1:1,000, o/n at 4°C 1:2,500 Open in a separate window Cul3, cullin 3; GST, glutathione S-transferase; HA, hemagglutinin; JAB1, jun activation domain-binding protein-1; Keap1, kelch-like ECH-associated protein 1; NEDD8, neuronal precursor cell expressed developmentally downregulated protein 8; Nrf2, nuclear factor erythroid 2-related factor 2; o/n, overnight; RT, room temperature.

Techniques: Inhibition, Western Blot, Activation Assay, Binding Assay

Journal: Diabetology & Metabolic Syndrome

Article Title: Effect of recurrent severe insulin-induced hypoglycemia on the cognitive function and brain oxidative status in the rats

doi: 10.1186/s13098-024-01410-z

Figure Lengend Snippet: GFAP-positive astrocytes immunostained with rabbit anti‐GFAP polyclonal antibody (400x magnification). The impact of severe recurrent hypoglycemia on GFAP expression, serving as a marker for astrocyte development, has been demonstrated in both the brain cortex and hippocampus. Tissue samples were collected from the control group (10 rats) and from each treatment group (14 rats) at 7 days and 90 days after treatment. Three tissue sections of the cortex and three sections of the hippocampus were taken from each animal. The distance between the sections was 30 μm. ( A ) Representative immunohistochemical images showcasing GFAP expression in the brain and hippocampus of the control rats and rats subjected to 90 min hypoglycemia for three times, at 7 days (Hypoglycemia, Short) and 3 months (Hypoglycemia, Long) after induction of hypoglycemia ( B ). Semi-quantitative evalution of GFAP‐labeled astrocytes in the brain cortex and hippocampus. The results demonstrated rats subjected to hypoglycemia showed significant evidence of astrocytosis development. The severity of astrocytosis three months after inducing hypoglycemia (Hypoglycemia, Long) was significantly reduced compared to seven days after inducing hypoglycemia (Hypoglycemia, Short). Significance levels are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Sections were then incubated in rabbit anti-GFAP (1:2.000; Z 334, DAKO), overnight, followed by rabbit anti-rat IgG conjugated to horseradish peroxidase (1:100; P 450, DAKO).

Techniques: Expressing, Marker, Control, Immunohistochemical staining, Labeling