1 ap  (Proteintech)

Journal: Cell calcium

Article Title: Differential modulation of SK channel subtypes by phosphorylation.

doi: 10.1016/j.ceca.2020.102346

Figure Lengend Snippet: Fig. 1. The apparent Ca2+ sensitivity of SK1 and IK channel subtypes is more decreased than that of the SK3 and SK2 subtypes in immortal ized endothelial cells. Concentration-dependent activation by Ca2+ of the SK channel subtypes heterologously expressed in HEK293 cells (A) and immortalized endothelial cells (B). (C) EC50 values for the activation by Ca2+, recorded from SK channel subtypes expressed in HEK293 cells (HEK) and immortalized endothelial cells (ET). All data are shown as mean ± S.E.M (n = 6-11, **P < 0.01, ***P < 0.001, Two-way ANOVA followed by Tukey’s post hoc tests).

Article Snippet: We isolated the SK2 channel protein from the plasma membrane of the cultured endothelial cells stably expressing SK2 channels through immunoprecipitation using GFP-Trap_A beads (Chromotek).

Techniques: Concentration Assay, Activation Assay

Journal: Cell calcium

Article Title: Differential modulation of SK channel subtypes by phosphorylation.

doi: 10.1016/j.ceca.2020.102346

Figure Lengend Snippet: Fig. 3. Subcellular localization of the SK channel subtypes in immortalized endothelial cells. (A) Cell fractions were verified by immunoblots with antibodies for mitochondria marker (Cytochrome C), ER marker (GRP-78) and plasma membrane marker (Na+-K+ ATPase). In (B), (C) and (D), densitometry results for the subcellular markers are summarized from 3-4 experiments. (E) In cell fractions, the SK2 channel subtype is abundantly expressed on the ER membrane, in contrast to other channel subtypes. In (F), (G), (H) and (I), densitometry results for the SK channel subtypes are summarized from 4-6 experiments. All data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001, Two-way ANOVA followed by Tukey’s post hoc tests.

Article Snippet: We isolated the SK2 channel protein from the plasma membrane of the cultured endothelial cells stably expressing SK2 channels through immunoprecipitation using GFP-Trap_A beads (Chromotek).

Techniques: Western Blot, Marker, Clinical Proteomics, Membrane

Journal: Cell calcium

Article Title: Differential modulation of SK channel subtypes by phosphorylation.

doi: 10.1016/j.ceca.2020.102346

Figure Lengend Snippet: Fig. 4. The SK2 channel subtype protects the immortalized endothelial cells against apoptosis induced by palmitate. (A) Represen tative flow cytometric analysis of non-treated cells and cells treated with 80 μM palmitate using double staining with Annexin V-Alexa Fluor 647/PI. Cells in the lower right quarter indicate AnnexinV-positive, early apoptotic cells. Cells in the upper right quarter indicate AnnexinV-positive/PI-positive, late apoptotic cells. (B) Quantification of cell death (the total of early and late apoptotic cells) after 24 h treatment with palmitate (mean ± S.E.M, n = 5; **P < 0.01, ***P < 0.001 compared with con trol parent cells, Two-way ANOVA followed by Tukey’s post hoc tests).

Article Snippet: We isolated the SK2 channel protein from the plasma membrane of the cultured endothelial cells stably expressing SK2 channels through immunoprecipitation using GFP-Trap_A beads (Chromotek).

Techniques: Double Staining

Journal: Cell calcium

Article Title: Differential modulation of SK channel subtypes by phosphorylation.

doi: 10.1016/j.ceca.2020.102346

Figure Lengend Snippet: Fig. 6. SK channels are phosphorylated by kinases. CK2 phosphorylates the CaM in complex with SK channels and leads to reduced apparent Ca2+ sensi tivity. PKA phosphorylates the C-terminus of SK2 channels and reduces its cell surface expression. Counter-regulatory phosphatases are not shown for the purpose of clarity.

Article Snippet: We isolated the SK2 channel protein from the plasma membrane of the cultured endothelial cells stably expressing SK2 channels through immunoprecipitation using GFP-Trap_A beads (Chromotek).

Techniques: Expressing

Journal: Cell calcium

Article Title: Differential modulation of SK channel subtypes by phosphorylation.

doi: 10.1016/j.ceca.2020.102346

Figure Lengend Snippet: Fig. 5. Nano-LC/MS/MS analyses of SK2 pro tein isolated from the plasma membrane of the immortalized endothelial cells. (A). The endo thelial cells expressing SK2 channels were sub jected to cell fractionation. SK2 channel proteins were purified from the plasma mem brane fraction and then separated on SDS-PAGE gel. Protein bands at ~50 kDa and ~17 kDa correspond to SK2 channels (labeled in red box) and calmodulin, respectively. (B). The extrac ted ion-chromatograms of the peptide “567RSSSTAPPTSSESS580” of SK2 channels (doubly charged ion at m/z 370.80457). (C). The tandem mass spectrum of the peptide was acquired from the doubly charged precursor ion at m/z 370.80457. Fragment ion peaks as b- or y-type ions are also labeled in the spectrum. The peptide sequence including the phosphor ylated sites at serines (red color) is indicated at the bottom.

Article Snippet: We isolated the SK2 channel protein from the plasma membrane of the cultured endothelial cells stably expressing SK2 channels through immunoprecipitation using GFP-Trap_A beads (Chromotek).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Clinical Proteomics, Membrane, Expressing, Cell Fractionation, Purification, SDS Page, Labeling, Sequencing