anti serca2 rabbit monoclonal antibody  (Cell Signaling Technology Inc)

Journal: Molecular metabolism

Article Title: Regulation of beige adipocyte thermogenesis by the cold-repressed ER protein NNAT.

doi: 10.1016/j.molmet.2023.101679

Figure Lengend Snippet: Figure 4: NNAT regulates calcium cycling and thermogenic respiration via SERCA2 (A) SERCA2 protein levels during differentiation of primary iWAT cells to beige adipocytes. (B and C) Immunoblot of SERCA2 and NNAT after immunoprecipitation by SERCA2 or control IgG in non-crosslinked iWAT cell lysate (B) or crosslinked iWAT lysate (C). For (C), two same sample sets were loaded in different lanes and one was probed with SERCA2 monoclonal antibody and the other with NNAT monoclonal antibody above 100 kDa. (D) Change in the intracellular calcium levels in WT and Nnat KO iWAT cells after treatment with 2 mM of thapsigargin (TG). WT, n ¼ 8; Nnat KO, n ¼ 9.(E) Change in the intracellular calcium levels in LacZ control and Nnat OE iWAT cells after treatment with 2 mM of TG. LacZ, n ¼ 5; Nnat OE, n ¼ 5. (F) Protein expression of Ryanodine receptor 1 (RYR1), Ryanodine receptor 2 (RYR2), and IP3 receptor isoform 1 (IP3R1) in the stromal vascular fraction (SVF) of iWAT, differentiated iWAT cells, and indicated tissues. (G) Change in the intracellular calcium levels in WT and Nnat KO iWAT cells after treatment with 50 mM of 2-aminoethyl diphenylborinate (2-APB). WT, n ¼ 10; Nnat KO, n ¼ 10. (H) Change in the intracellular calcium levels in LacZ control and Nnat OE iWAT cells after treatment with 50 mM of 2-APB. LacZ, n ¼ 6; Nnat OE, n ¼ 6. (I and J) OCR in WT and Nnat KO iWAT cells treated with 2 mM TG or 50 mM 2-APB for 30 min in the absence (I) or presence of 10 mM CL316,243 (J). At least biological triplicates were used. P-value is determined by two-tailed Student’s t-test (I, J) or two-way ANOVA followed by Fisher’s LSD test (D, E, G, H).

Article Snippet: We used NNAT monoclonal antibody (Abcam, 1:3000 dilution), NHLRC1 polyclonal antibody (Fisher Scientific, 1:1000), NHLRC1 monoclonal antibody (Fisher Scientific, 1:1000), SERCA2 monoclonal antibody (Cell Signaling, 1:1000), RYR1 monoclonal antibody (Fisher Scientific, 1:1000), RYR2 monoclonal antibody (Sigma, 1:1000), IP3R1 monoclonal antibody (Cell Signaling, 1:1000), UCP1 monoclonal antibody (R&D systems, 1:3000), FABP4 monoclonal antibody (Santa Cruz, 1:3000), and b-actin monoclonal antibody (Santa Cruz, 1:1000).

Techniques: Western Blot, Immunoprecipitation, Control, Expressing, Two Tailed Test

Journal: Molecular metabolism

Article Title: Regulation of beige adipocyte thermogenesis by the cold-repressed ER protein NNAT.

doi: 10.1016/j.molmet.2023.101679

Figure Lengend Snippet: Figure 6: Overview of functional and physical interaction between NNAT and SERCA. A schematic model of regulation of noncanonical thermogenesis by NNAT in adipocytes. At thermoneutrality, NNAT interacts with SERCA2 and inhibits calcium cycling. Cold exposure or treatment with a b3-adrenergic agonist increases intracellular cAMP levels, which induces NHLRC1 expression. The E3 ubiquitin ligase NHLRC1 then ubiquitinates and degrades NNAT, leading to activation of SERCA2. The futile calcium cycling mediated by SERCA2 dissipates energy as heat.

Article Snippet: We used NNAT monoclonal antibody (Abcam, 1:3000 dilution), NHLRC1 polyclonal antibody (Fisher Scientific, 1:1000), NHLRC1 monoclonal antibody (Fisher Scientific, 1:1000), SERCA2 monoclonal antibody (Cell Signaling, 1:1000), RYR1 monoclonal antibody (Fisher Scientific, 1:1000), RYR2 monoclonal antibody (Sigma, 1:1000), IP3R1 monoclonal antibody (Cell Signaling, 1:1000), UCP1 monoclonal antibody (R&D systems, 1:3000), FABP4 monoclonal antibody (Santa Cruz, 1:3000), and b-actin monoclonal antibody (Santa Cruz, 1:1000).

Techniques: Functional Assay, Expressing, Ubiquitin Proteomics, Activation Assay