Journal: Cell reports
Article Title: PKCε Inhibits Neuronal Dendritic Spine Development through Dual Phosphorylation of Ephexin5
doi: 10.1016/j.celrep.2018.11.005
Figure Lengend Snippet: (A) Protein kinase C (PKC) isoform expression is detectable in the developing brain and increases with age. Mouse brains were dissected and lysed at indicated ages and immunoblotted with isoform specific PKC antibodies. (B) PKC isoforms are brain enriched. Lysates from indicated mouse tissues (Br, brain; Ht, heart; Kd, kidney; Li, liver) at postnatal day 6 (P6) were immunoblotted for the PKC isoforms shown. Equal protein loading was confirmed using Silver Stain. (C) PKC isoforms are expressed in the hippocampus early in development.Lysates were prepared from cerebral cortex (Ctx), hippocampus (Hip), diencephalon/midbrain/hindbrain (DMH), and olfactory bulbs (OB) from P6 mice and immunoblotted for the PKC isoforms shown. (D) Experimental setup. Fractionation protocol for collecting samples tomeasure PKC membrane association. (E) PKC isoforms are localized at neuronal membranes in the hippocampusearly in development, indicative of their activation. Fractionated extracts generated from neonatal mouse hippocampi were immunoblotted for the PKC isoforms shown. Cyto, cytosolic proteins co-fractionating with Tuj1; Mem, membrane-associated proteins co-fractionating with transferrin receptor (TFRC); Insol, remaining fraction of detergent-insoluble proteins. (F) PKC isoform expression in hippocampal neuronal cultures increases withage of the culture. Cultured hippocampal neurons were lysed at indicated days in vitro (DIV) and immunoblotted for the PKC isoforms shown. (G) PKCa, PKCb, and PKCg are localized at neuronal membranes in immature hippocampal neuronal cultures. PKCε membrane localization is scarce by comparison. Fractionated extracts of DIV11 neuronal cultures were prepared as in (D) and immunoblotted. See also .
Article Snippet: The following antibodies are commercially available and used according to manufacturer’s suggestions for Western Blots, immunocytochemistry and immunoprecipitation: PKCa (BD Biosciences 610107), PKCb (BD Biosciences 610127), PKCg (Santa Cruz Biotechnology SC-211), PKCε (BD Biosciences 610085), RhoA (Santa Cruz Biotechnology SC-418), b-Actin (Abcam ab8226), Tuj1/bIII-tubulin (EMD Millipore TU-20), Transferrin receptor (Invitrogen 136800), HA (Sigma H3663), Myc (Abcam ab32), Synapsin (Millipore AB1543P), Synaptophysin (Millipore MAB368), PSD95 (Thermo Scientific MA1–046), and GAPDH ( ).
Techniques: Expressing, Silver Staining, Fractionation, Activation Assay, Generated, Cell Culture, In Vitro
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