Journal: Cell reports

Article Title: Retrograde adenosine/A 2A receptor signaling facilitates excitatory synaptic transmission and seizures

doi: 10.1016/j.celrep.2024.114382

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PKI 6-22 amide , Tocris Bioscience , Cat#1904.

Techniques: Virus, Plasmid Preparation, Recombinant, Software

Journal: eLife

Article Title: Renal interstitial cells promote nephron regeneration by secreting prostaglandin E2

doi: 10.7554/eLife.81438

Figure Lengend Snippet: ( A–C ) lhx1a mRNA levels were assessed by RT-PCR at 7 dpi. β-actin was used as a sample control. ( D–K ) lhx1a whole-mount in situ hybridization (WISH) showing the trunk kidney region at 7 dpi. XAV939 ( F ) could reduce the number of lhx1a + cell aggregates and dmPGE2 could rescue the influence of XAV939 treatment ( G ); ICRT 14 ( H ) or ICG-001 ( J ) could reduce the number of lhx1a + cell aggregates and dmPGE2 could not rescue the influence of ICRT 14 ( I ) or ICG-001 ( K ) treatment. n = 5–7 in each condition. ( L ) lhx1a + cell aggregates of whole kidney were calculated using ImageJ. Data were analyzed by ANOVA, ***p<0.001; ns, no significant difference. ( M–V ) Immunofluorescence staining of β-catenin in Tg(lhx1a:DsRed ) zebrafish kidneys at 5 dpi. ( M, N ) Zebrafish injected DMSO as a control group, and the amount of β-catenin could be detected in lhx1a + cell aggregates during renal progenitor cell (RPC) aggregation ( M ) or proliferation ( N ). ( O ) β-catenin level in lhx1a + cell aggregates of cox2a -/- was significantly less than the control group, and injection of dmPGE2 ( P, Q ) could rescue the influence of Cox2a deficiency. ( R ) β-catenin level in lhx1a + cell aggregates of wnt4a -/- was significantly less than the control group, and injection of dmPGE2 ( S, T ) could rescue the influence of Wnt4a deficiency. Injection of PKI ( U ) could reduce β-catenin level in lhx1a + cell aggregates, while injection of dmPGE2 ( V ) could not rescue the influence of PKI treatment. n = 3–6 in ( M–V ). Scale bar in ( M–V ), 50 μm. ( W ) Bar chart depicting β-catenin levels following acute kidney injury (AKI) ( M–V ). Fluorescent intensities per unit area were measured at the lhx1a + RPC aggregates using ImageJ. β-catenin levels of lhx1a + RPCs during RPC aggregation normalized as 1. Ag, aggregation; Pr, proliferation. n = 3–6 in each condition. Data were analyzed by ANOVA, ***p<0.001; ns, no significant difference. Figure 6—source data 1. Original gel files of . Figure 6—source data 2. Numerical data for , , , and .

Article Snippet: During the nephron regeneration stage, the Indo (I7378-5G, Sigma; 400 μM, 10 μL per fish), NS-398 (N194-5MG, Sigma; 140 μM, 10 μL per fish), TG4-155 (S6793, Selleck; 400 μM, 10 μL per fish), dmPGE2 (D0160, Sigma; 600 μM, 10 μL per fish), GW627368X (T1978, TOPSCIENCE; 200 μM, 10 μL per fish), XAV939 (S1180, Selleck; 200 μM, 10 μL per fish), ICRT 14 (HY-16665, MCE; 10 μM, 10 μL per fish), ICG-001 (HY-14428, MCE; 100 μM, 10 μL per fish), PKI (HY-P1290A, MCE; 20 μM, 10 μL per fish), and H89 (HY-15979, MCE; 200 μM, 10 μL per fish) were intraperitoneally injected into zebrafish at 2, 4, and 6 dpi.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, In Situ Hybridization, Immunofluorescence, Staining, Injection

Journal: eLife

Article Title: Renal interstitial cells promote nephron regeneration by secreting prostaglandin E2

doi: 10.7554/eLife.81438

Figure Lengend Snippet: ( A, B ) lhx1a mRNA levels were evaluated by RT-PCR at 7 dpi. β-actin was used as a sample control. ( C–H ) lhx1a whole-mount in situ hybridization (WISH) showing the trunk kidney region at 7 dpi. PKI ( E ) or H89 ( G ) treatment reduced the number of lhx1a + cell aggregates, while injection of dmPGE2 could not rescue the influence of PKI ( F ) or H89 ( H ) treatment. ( I ) lhx1a + cell aggregates of whole kidney were calculated using ImageJ. n = 5–7 in each condition. Data were analyzed by ANOVA, ***p<0.001; ns, no significant difference. ( J–N ) Immunofluorescence staining of p-S9-GSK3β (arrowheads) in Tg(lhx1a:DsRed ) zebrafish kidneys at 5 dpi. ( J, K ) Zebrafish injected with DMSO as a control group, and the amount of p-S9-GSK3β could be detected in lhx1a + cell aggregates cytoplasm during RPC aggregation ( J ) or proliferation ( K ). ( L ) p-S9-GSK3β in lhx1a + cell aggregates of cox2a -/- was hardly detectable, and injection of dmPGE2 ( M, N ) could rescue the influence of Cox2a deficiency. Injection of PKI ( O ) could reduce p-S9-GSK3β level in lhx1a + cell aggregates, while injection of dmPGE2 ( P ) could not rescue the influence of PKI treatment. ( Q–W ) Immunofluorescence staining of p-S675-β-catenin in Tg(lhx1a:DsRed ) zebrafish kidneys at 5 dpi. ( Q, R ) Injection of DMSO as a control group and amounts of p-S675-β-catenin could be detected in lhx1a + cell aggregates during RPC aggregation ( Q ) or proliferation ( R ). ( S ) p-S675-β-catenin level in lhx1a + cell aggregates of cox2a -/- was hardly detectable, and injection of dmPGE2 ( T, U ) could rescue the influence of Cox2a deficiency. Injection of PKI ( V ) could reduce p-S675-β-catenin level in lhx1a + cell aggregates, while injection of dmPGE2 ( W ) could not rescue the influence of PKI treatment. Scale bar, 50 μm. ( X, Y ) Bar chart depicting p-S9-GSK3β ( X ) and p-S675-β-catenin ( Y ) levels following acute kidney injury (AKI) ( J–W ). Fluorescent intensities per unit area were measured at the lhx1a + RPC aggregates using ImageJ. p-S9-GSK3β or p-S675-β-catenin levels of lhx1a + RPCs during RPC aggregation normalized as 1. Ag, aggregation; Pr, proliferation. n = 3–6 in each condition. Data were analyzed by ANOVA, ***p<0.001; ns, no significant difference. Figure 7—source data 1. Original gel files of . Figure 7—source data 2. Numerical data for .

Article Snippet: During the nephron regeneration stage, the Indo (I7378-5G, Sigma; 400 μM, 10 μL per fish), NS-398 (N194-5MG, Sigma; 140 μM, 10 μL per fish), TG4-155 (S6793, Selleck; 400 μM, 10 μL per fish), dmPGE2 (D0160, Sigma; 600 μM, 10 μL per fish), GW627368X (T1978, TOPSCIENCE; 200 μM, 10 μL per fish), XAV939 (S1180, Selleck; 200 μM, 10 μL per fish), ICRT 14 (HY-16665, MCE; 10 μM, 10 μL per fish), ICG-001 (HY-14428, MCE; 100 μM, 10 μL per fish), PKI (HY-P1290A, MCE; 20 μM, 10 μL per fish), and H89 (HY-15979, MCE; 200 μM, 10 μL per fish) were intraperitoneally injected into zebrafish at 2, 4, and 6 dpi.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, In Situ Hybridization, Injection, Immunofluorescence, Staining

Journal: BMC Molecular Biology

Article Title: A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability

doi: 10.1186/1471-2199-9-22

Figure Lengend Snippet: S. cerevisiae Sup35p is phosphorylated by PKA in vitro . Proteins were incubated with the PKA catalytic subunit and [γ- 32 P]ATP, submitted to SDS-PAGE, and visualized by autoradiography. (a) Phosphorylation of wild-type Sup35p in the presence of PKA or PKI. (b) Phosphorylation of A and D mutant proteins by PKA. (c) Recognition of the different proteins by a specific PKA-phosphorylated substrate antibody. Wild-type, A or D mutant proteins were phosphorylated or not by PKA before western blotting and detection using a secondary antibody linked to the alkaline phosphatase. (d) Phosphorylation of C-terminal proteins by PKA.

Article Snippet: To test for the specificity of the reaction for PKA, 30 μg of PKA inhibitor (PKI) (Sigma) were added to the reaction buffer to specifically inhibit the activity of PKA.

Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Mutagenesis, Western Blot