Journal: PLoS Pathogens

Article Title: Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase

doi: 10.1371/journal.ppat.1004768

Figure Lengend Snippet: (A) 293T cells were transfected with a plasmid containing kGPCR. kGPCR was purified with anti-Flag affinity chromatography. kGPCR-interacting proteins were analyzed by commassie staining and photographed, identified by mass spectrometry analysis. A separating line indicates a lane that was removed between lanes of vector and kGPCR. Note, heat-denatured kGPCR oligomerized and migrated slowly. (B) 293T cells were transfected with plasmids containing SERCA2 or kGPCR. Centrifuged cell extract was precipitated with anti-HA (kGPCR). Precipitated proteins and whole cell lysates were analyzed by immunoblot with indicated antibodies. (C and D) HUVEC endothelial stable cells were analyzed by proximity ligation assay (red) and stained with DAPI. Cells were analyzed by confocal microscopy (C). The intensity of proximity ligation was semi-quantitatively determined by counting more than 100 cells (D). (E) 293T cells were transfected with a plasmid containing Flag-tagged SERCA2 without or with a plasmid containing kGPCR. SERCA2 was precipitated with anti-Flag agarose and analyzed by ATPase activity in vitro. Precipitated SERCA2 and whole cell lysates were analyzed by immunoblot (insert). (F) 293T cells were transfected with an NFAT reporter cocktail, a plasmid containing kGPCR and increasing amount of a plasmid containing SERCA2. NFAT activation was determined by luciferase reporter assays.

Article Snippet: Commercial antibodies used in this study include mouse anti-HA monoclonal antibody and agarose (Sigma), mouse anti- β-Actin monoclonal antibody (Abcam), rabbit anti-COX-2 polyclonal antibody (Abcam), rabbit anti-RCAN1 polyclonal antibody (Sigma), mouse anti-SERCA2 (IID8) monoclonal antibody (Santa cruz), mouse anti-calcineurin Aα (Santa cruz).

Techniques: Transfection, Plasmid Preparation, Purification, Affinity Chromatography, Staining, Mass Spectrometry, Western Blot, Proximity Ligation Assay, Confocal Microscopy, Ligation, Activity Assay, In Vitro, Activation Assay, Luciferase

Journal: Circulation Research

Article Title: Endothelial S100A1 Modulates Vascular Function via Nitric Oxide

doi: 10.1161/circresaha.108.172031

Figure Lengend Snippet: Figure 6. S100A1 colocalizes with both SERCA2 and the IP3R. A, C, D, and F, Representative immunolabeling of SERCA2 (red) (A), IP3R (red) (D), and S100A1 (green) (C and F). Overlays of A and C (B) and D and F (E) reveal similar intracellular localization of both SERCA2 and IP3R with S100A1 protein. Original magnifi- cation, 60.

Article Snippet: Co- immunofluorescence for S100A1 and sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2) Online Supplement Pleger et al., Endothelial S100A1 Modulates Vascular... 2 as well as for S100A1 and the inositol 1,4,5- triphosphate receptor (IP3R) was performed in RCEC using the S100A1 AB as described above and, consecutively, a goat-anti SERCA2 (sc- 8095, 1:100) or a goat-anti IP3R AB (sc-7278, Santa Cruz, CA) followed by consecutive probing with a 488 donkey anti-rabbit and a 555 donkey anti-goat AB’s (Molecular Probes; 1:100).

Techniques: Immunolabeling

Journal: PLoS ONE

Article Title: Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

doi: 10.1371/journal.pone.0058128

Figure Lengend Snippet: Real-time PCR analysis showing SERCA2 (A), RyR2 (B), IP3-RI (C) and IP3-RIII mRNA expression levels in HL-1 cardiomyocytes exposed to increasing doses of LDL. Data were processed with a specially designed software programme based on Ct value of each sample and normalized to ARBP mRNA. Values significantly different from the level with 0 µg/ml LDL are indicated with asterisks.

Article Snippet: Blots were incubated with monoclonal antibodies against SERCA2 (Novus Biologicals NB100–237), Cx40 (Invitrogen, 36–4900) and Cx43 (Sigma-Aldrich C6219).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Software

Journal: PLoS ONE

Article Title: Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

doi: 10.1371/journal.pone.0058128

Figure Lengend Snippet: Representative Western blot analysis showing SERCA2 bands in HL-1 cells exposed to increasing LDL concentrations (0, 50, 100 and 200 µg/mL) ( A ) or to similar dose (500 µg/ml) of LDL or HDL ( B ) for 24 hours. The bar graph below shows SERCA2 band quantification. Unchanged levels of β-tubulin are shown as loading control. Results are mean±SEM of three independent experiments performed in duplicate. *P<0.05 versus. HL-1 cells incubated in absence of LDL. C Calcium images acquired at rest before (CON) and during a rapid caffeine application. D Representative calcium transients recorded in cultures incubated without lipoproteins (CON), with 500 µg/ml LDL or with 500 µg/ml HDL. The lower panel shows the corresponding time integrals of the calcium transients. E summary of the effect of LDL and HDL on the time integral of the caffeine induced calcium transient. *P<0.05 vs. cells incubated with LDL

Article Snippet: Blots were incubated with monoclonal antibodies against SERCA2 (Novus Biologicals NB100–237), Cx40 (Invitrogen, 36–4900) and Cx43 (Sigma-Aldrich C6219).

Techniques: Western Blot, Control, Incubation