Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

doi: 10.1159/000495229

Figure Lengend Snippet: Fig. 2. Effect of lithium treatment without and with NFκB inhibitor wogonin on Orai1, STIM1 and STIM2 protein abundance in iPSC-derived neurons from ChAc patients. A,B,C. Original Western blot of (A) Orai1, (B) STIM1 and (C) STIM2 protein abundance in neurons differentiated from iPSCs derived from ChAc patients (ChAc) without or with lithium (24 h, 2 mM) treatment without and with presence of NFκB inhibitor wogonin (50 µM). D,E,F. Arithmetic means (± SEM, n = 5) of (D) Orai1, (E) STIM1 and (F) STIM2 protein abundance in neurons generated from iPSCs derived from ChAc patients without (blue bars) or with (red and grey bars) lithium (24 h, 2 mM) treatment without (red bars) and with (grey bars) presence of NFκB inhibitor wogonin (50 µM). #(p<0.05), ##(p<0.01) indicates statistically significant difference to respective value in the absence of treatment, §(p<0.05), §§§(p<0.001) indicates statistically significant difference to respective value in the absence of wogonin.

Article Snippet: The proteins were separated by 10% SDS-PAGE in Glycine-Tris buffer and electro-transferred onto nitrocellulose membranes for 90 min. After blocking with 5% milk in TBST at room temperature for 1 h, the membranes were incubated with primary anti-ORAI1 antibody (1:1000, Proteintech), anti-STIM1 antibody (1:1000, Cell Signaling), anti-STIM2 antibody (1:1000, Cell Signaling) and anti-GAPDH antibody (1:1000, Cell Signaling) at 4°C overnight.

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synaptic crosstalk conferred by a zone of differentially regulated Ca 2+ signaling in the dendritic shaft adjoining a potentiated spine

doi: 10.1073/pnas.1902461116

Figure Lengend Snippet: LTCC activity level governs the spread along the dendritic shaft of STIM1 feedback inhibition of dendritic LTCCs. (A) Mean total cytosolic Ca2+ (RGECO1) changes by uncaging on 79wt neurons at 0 μm. Mean (black) ± SEM (dark gray). Uncaging occurred from 0 to 60 s, at 1 Hz. (B) Mean LTCC-independent Ca2+ changes (IF) in the cytosol by uncaging at 0 μm in the same neurons as A and pretreated with nimodipine (5 μM) for 15 min. Mean (black) ± SEM (red). (C) Mean LTCC-dependent Ca2+ changes (DF) at 0 μm, calculated by subtracting the non-LTCC Ca2+ time course from the total Ca2+ time course of the same neuron. Mean (black) ± SEM (blue). (D) Mean integrated increase in total cytosolic Ca2+ (shaded gray region in A) along a dendrite of 79wt (circles; n = 5), 79ΔPIX (squares; n = 5), and 79ΔPKA (diamonds; n = 6) neurons. Mean ± SEM. (E) Mean integrated increase in the LTCC-independent component of cytosolic Ca2+ (IF; shaded pink region in B) along a dendrite of 79wt (circles; n = 5), 79ΔPIX (squares; n = 5), and 79ΔPKA (diamonds; n = 6) neurons. Mean ± SEM. (F) Mean integrated increase in LTCC-dependent component of cytosolic Ca2+ (DF; shaded light blue region in C) along a dendrite of 79wt (circles; n = 5), 79ΔPIX (squares; n = 5), and 79ΔPKA (diamonds; n = 6) neurons. Mean ± SEM.

Article Snippet: D1ER (Addgene no. 36325), RGECO1 (Addgene no. 32444), and human STIM1-YFP (Addgene no. 19754) were provided by Amy Palmer, University of Colorado, Boulder, CO, Robert Campbell, University of Alberta, Edmonton, Canada, and Anjana Rao, La Jolla Institute for Immunology, La Jolla, CA (via Addgene).

Techniques: Activity Assay, Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synaptic crosstalk conferred by a zone of differentially regulated Ca 2+ signaling in the dendritic shaft adjoining a potentiated spine

doi: 10.1073/pnas.1902461116

Figure Lengend Snippet: β-adrenergic receptors modulate a zone of Ca2+ signaling in the dendritic shaft adjoining a potentiated spine. (A) In neurons treated with Iso, time course of change in total cytosolic Ca2+ (IF + DF, imaged with RGECO1) (n = 5) measured at 0 μm from the uncaging-stimulated spine. Mean (black) ± SEM (dark gray). (B) After 15 min nimodipine treatment, time course of change in LTCC-independent cytosolic Ca2+ (IF) of the neurons in A, measured at 0 μm. Mean (black) ± SEM (red). (C) Time course of increase in LTCC-dependent cytosolic Ca2+ (DF) (DF = total – IF) at 0 μm. Mean (black) ± SEM (blue). (D) Spatial profile for integrated increase in total cytosolic Ca2+ (IF + DF) shaded gray region in A along the dendrite of Iso-treated (1 μM; open circles; n = 5) versus untreated (control) neurons (shaded circles; n = 5) and for Iso-treated + STIM1 knockdown (squares; n = 5). Mean ± SEM. (E) Spatial profile for integrated increase in the LTCC-independent Ca2+ (IF; shaded pink region in B) along the dendrite of Iso-treated (open circles; n = 5) versus untreated (control) neurons (red circles; n = 5) and for Iso-treated + STIM1 knockdown (squares; n = 5). Mean ± SEM. (F) Spatial profile for average integrated increase in LTCC-dependent Ca2+ (DF; shaded light blue region in C) along the dendrite of Iso-treated (open circles; n = 5) versus untreated (control) neurons (blue circles; n = 5) and for Iso-treated + STIM1 knockdown (squares; n = 5). Mean ± SEM.

Article Snippet: D1ER (Addgene no. 36325), RGECO1 (Addgene no. 32444), and human STIM1-YFP (Addgene no. 19754) were provided by Amy Palmer, University of Colorado, Boulder, CO, Robert Campbell, University of Alberta, Edmonton, Canada, and Anjana Rao, La Jolla Institute for Immunology, La Jolla, CA (via Addgene).

Techniques: Control, Knockdown