golgi apparatus enrichment kit  (Invent Biotechnologies)

Journal: iScience

Article Title: PKA orchestrates long-range lysosomal vesicle transport during synaptic maintenance

doi: 10.1016/j.isci.2025.113878

Figure Lengend Snippet: PKA signaling modulates the transport of LVs during synapse maintenance (A) Experiment design of electrophysiological recordings before and 30 mins after PKA inhibitor (PKAi) application. (B) Representative trace of SN-L7MN (DIV 4–6) excitatory postsynaptic potentials (EPSPs) before (pink) and after (purple) 14–22 amide (+PKAi) in millivolts (mV); scale display 1 mV along the y axis and 0.1 s (s) in the x axis. (C) Bar graph shows average EPSPs in mV of SN-L7MN before (pre) and 30 min after +PKAi application. The number of neurons analyzed in the experiment is indicated in gray. Error bars show SEMs. ∗∗ p < 0.001. Student’s paired t test. (D) Experiment design. (E and F) Bar graphs show the flux and velocity of retrograde LV (E) and bidirectional mitochondrial (F) transport before and after PKA inhibition with 14–22 amide (PKAi) analyzed from kymographs, respectively. The number of neurons analyzed in the experiment is indicated in gray. Student’s paired t test (LV flux, mitochondrial flux); Student’s unpaired two-tailed t test (LV velocity and mitochondrial velocity). N in graph indicates number of biological replicates and n in graph indicates number of technical replicates. (G) Workflow describing the isolation of Golgi enriched fractions from Aplysia neuronal lysate. Aplysia nervous system was isolated from the control and LTS trained animals and homogenized in Golgi extraction buffer (Minute Golgi Enrichment Kit, Invent biotechnologies). The fractions obtained by differential centrifugation, were analyzed by western blot to confirm the Golgi enrichment using GM130 Ab, which detects cis Golgi matrix protein GM130. (H) Representative western blot image of total Aplysia lysate detecting phosphorylated and total PKA in control and LTS samples. The bar graph shows a quantitative representation (ImageJ) of western blot data showing the ratio of phosphoPKA to total PKA ( n = 5) which increases significantly in the LTS samples compared to the controls ( n = 5, p value = 0.0036). (I) Western blot images of the Golgi-enriched fractions along with total lysate. The fractions isolated by this protocol showed an enrichment of Golgi matrix protein compared to the total lysate as shown by the GM130 protein. Phosphorylated PKA is found less in Golgi enriched fractions of LTS samples compared to the control samples. The bar graph shows quantitative representation of the ratio of phosphoPKA/total PKA in the Golgi-enriched fractions of LTS and control samples (ImageJ, n = 4, p value = 0.0015).

Article Snippet: Aplysia nervous system was isolated from the control and LTS trained animals and homogenized in Golgi extraction buffer (Minute Golgi Enrichment Kit, Invent biotechnologies).

Techniques: Inhibition, Two Tailed Test, Isolation, Control, Extraction, Centrifugation, Western Blot

Journal: iScience

Article Title: PKA orchestrates long-range lysosomal vesicle transport during synaptic maintenance

doi: 10.1016/j.isci.2025.113878

Figure Lengend Snippet: PKA signaling modulates the transport of LVs during synapse maintenance (A) Experiment design of electrophysiological recordings before and 30 mins after PKA inhibitor (PKAi) application. (B) Representative trace of SN-L7MN (DIV 4–6) excitatory postsynaptic potentials (EPSPs) before (pink) and after (purple) 14–22 amide (+PKAi) in millivolts (mV); scale display 1 mV along the y axis and 0.1 s (s) in the x axis. (C) Bar graph shows average EPSPs in mV of SN-L7MN before (pre) and 30 min after +PKAi application. The number of neurons analyzed in the experiment is indicated in gray. Error bars show SEMs. ∗∗ p < 0.001. Student’s paired t test. (D) Experiment design. (E and F) Bar graphs show the flux and velocity of retrograde LV (E) and bidirectional mitochondrial (F) transport before and after PKA inhibition with 14–22 amide (PKAi) analyzed from kymographs, respectively. The number of neurons analyzed in the experiment is indicated in gray. Student’s paired t test (LV flux, mitochondrial flux); Student’s unpaired two-tailed t test (LV velocity and mitochondrial velocity). N in graph indicates number of biological replicates and n in graph indicates number of technical replicates. (G) Workflow describing the isolation of Golgi enriched fractions from Aplysia neuronal lysate. Aplysia nervous system was isolated from the control and LTS trained animals and homogenized in Golgi extraction buffer (Minute Golgi Enrichment Kit, Invent biotechnologies). The fractions obtained by differential centrifugation, were analyzed by western blot to confirm the Golgi enrichment using GM130 Ab, which detects cis Golgi matrix protein GM130. (H) Representative western blot image of total Aplysia lysate detecting phosphorylated and total PKA in control and LTS samples. The bar graph shows a quantitative representation (ImageJ) of western blot data showing the ratio of phosphoPKA to total PKA ( n = 5) which increases significantly in the LTS samples compared to the controls ( n = 5, p value = 0.0036). (I) Western blot images of the Golgi-enriched fractions along with total lysate. The fractions isolated by this protocol showed an enrichment of Golgi matrix protein compared to the total lysate as shown by the GM130 protein. Phosphorylated PKA is found less in Golgi enriched fractions of LTS samples compared to the control samples. The bar graph shows quantitative representation of the ratio of phosphoPKA/total PKA in the Golgi-enriched fractions of LTS and control samples (ImageJ, n = 4, p value = 0.0015).

Article Snippet: Minute Golgi apparatus Enrichment Kit (20 tests) , Invent Biotechnologies , Cat#GO-037-IB.

Techniques: Inhibition, Two Tailed Test, Isolation, Control, Extraction, Centrifugation, Western Blot