cdk5 activator p35  (Addgene inc)

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: A. Single nucleus RNAseq data from hDRG showing the expression of CDK5 and CDK5R1 (encoding p35). Data are derived from Nguyen et al., 2021 . B. A human p35 expression vector was transfected into N2a cells. An empty vector (EV) and a His-tagged p35 vector were used as controls. Western blot for p35 was performed before sending the expression vector to be packaged into HSV. A 35 kDa band corresponding to the untagged human p35 was detected by Western. C. SH-SY5Y cells were differentiated and then transfected with a His-tagged p35 expression vector or an empty vector. Next, cells were treated with the following inflammatory mediators: Lane 1, non-treated; lane 2, 100nM bradykinin (BK) + 1µM PGE 2 (PG); lane 3, 10µM BK + 10µM PG; lane 4, 100nM BK + 1µM PG + 10µM 5HT + 10µM histamine; lane 5, 100nM BK + 1µM PG + 1mM 5HT + 1mM histamine; lane 6, 10µM BK + 10µM PG + 10µM 5HT + 10µM histamine; lane 7, 10µM BK + 10µM PG + 1mM 5HT + 1mM histamine. CDK5/p35 was immunoprecipitated and kinase activity was evaluated by the level of phosphorylated P histone H1, a substrate of cyclin-dependent kinases.

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Transfection, Western Blot, Immunoprecipitation, Activity Assay

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: A. Representative image showing the patched hDRG neurons under the inverted microscope. B. A representative AP trace ( black ) evoked at rheobase and a superimposed subthreshold trace without AP ( dashed orange trace ) in three groups (uninfected control; UI), (GFP control; GFP) and (p35 transfected; p35 hDRG neurons) before (baseline) and after perfusion with prostaglandin E2 (1μM) and bradykinin (100nM) ( red trace ). C. A representative enlarged AP trace depicting AP measurements. Rheobase: Minimal required threshold current to evoke an AP by an incrementing series of depolarizing pulse (incremental step; 50 pA and duration; 20 milliseconds), AP voltage threshold and resting membrane potential (RMP) are shown as dashed pink line and dashed red line respectively. AP rise time : AP rise to the peak, AP amplitude : AP peak height from RMP, AP overshoot: AP amplitude from zero membrane potential, AP fall time: AP peak back to the threshold value, AHP : (afterhyperpolarization) amplitude peak amplitude RMP. AP half width : AP duration at half the AP amplitude.

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques: Inverted Microscopy, Control, Transfection, Membrane

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: Bar diagrams (mean ± SEM) showing RMP (A), and rheobase current (B) in neurons from UI ( n=10; black circle ), GFP ( n=9; green circle ) and p35 ( n=9; orange circle ) groups. Statistical comparisons were performed using Kruskal Wallis test followed by Dunn’s multiple comparisons test. * p < 0.05, ns (non-significant).

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques:

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: Bar diagrams (mean ± SEM) showing AP rise time (A), AP fall time (B), AP half width (C), AP amplitude (D), AP overshoot (E), and AHP (F) in neurons from UI ( n=10; black circle ), GFP ( n=9; green circle ) and p35 ( n=9; orange circle ) groups. Statistical comparisons were performed using Kruskal Wallis test followed by Dunn’s multiple comparisons test. * p < 0.05, ** p < 0.01, ns (non-significant).

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques:

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: Paired dot plot graph depicting (A) RMP, and (B) rheobase current before (baseline; B) and after 1μM prostaglandin E2 and 100 nM bradykinin (PG/BK) in UI ( n=10; black circle ), GFP ( n=9; green circle ) and p35 ( n=9; orange circle ) neurons. In each section, the upper panel represents absolute value and lower panel represents % change. Statistical comparisons were performed using Wilcoxon signed rank test for paired data analysis and Kruskal Wallis test for percent changes. Dunn’s multiple comparisons test was performed for pairwise comparisons of percent changes. * p < 0.05, ** p < 0.01, ns (non-significant).

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques:

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: Paired dot plot graph depicting (A) AP rise time, (B) AP fall time, (C) AP half width, (D) AP amplitude, (E) AP overshot, (F) AHP before (baseline; B) and after 1μM prostaglandin E2 and 100 nM bradykinin (PG/BK) in UI ( n=10; black circle ), GFP ( n=9; green circle ) and p35 ( n=9; orange circle ) neurons. In each section, the upper panel represents absolute value and lower panel represents % change. Statistical comparisons were performed using Wilcoxon signed rank test for paired data analysis and Kruskal Wallis test for percent changes. Dunn’s multiple comparisons test was performed for pairwise comparisons of percent changes. * p < 0.05, ** p < 0.01, ns (non-significant).

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques: