Journal: Nature Communications
Article Title: Crbn modulates calcium influx by regulating Orai1 during efferocytosis
Figure Lengend Snippet: Crbn interacts with Orai1. a , b Phagocytes from the indicated mice a or Crbn deleted 293T cells by CRISPR/Cas9 b were lysed and proteins in the lysates were detected using immunoblotting. Data are representative of three independent experiments. c The transcript levels of Orai1 were analyzed by conventional PCR (bottom) or qRT-PCR (top) using cDNA synthesized from total mRNA extracted from the indicated BMDMs. n = 3 experiments, mean ± SEM. NS not significant (two-tailed unpaired Student t test). d LR73 cells were transfected with the indicated plasmid. At 1 day after transfection, the cells were lysed and proteins in the lysates were detected with the indicated antibodies. Data are representative of three independent experiments. e , f 293T cells transfected with the indicated plasmids e or BMDMs f were lysed and then the lysates were incubated with anti-FLAG antibody-conjugated agarose beads e or an anti-Orai1 antibody and protein A/G agarose beads f . Bead-bound proteins were detected with the indicated antibodies. Data are representative of five e or three f independent experiments. IP immunoprecipitation, TCL total cell lysates. g – i 293T cells were transfected with the indicated plasmids. At 2 days after transfection, the cells were lysed and the lysates were incubated with agarose beads conjugated with glutathione g , i or an anti-FLAG antibody h . Bead-bound proteins were detected with the indicated antibodies. Data are representative of four g or three h , i independent experiments. j Yeast cells transformed with the indicated plasmids were plated on selective or non-selective media. Cells on the non-selective media indicate the number of cells plated. BD binding domain, AD activation domain.
Article Snippet: Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.
Techniques: Mouse Assay, CRISPR, Polymerase Chain Reaction, Quantitative RT-PCR, Synthesized, Two Tailed Test, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Transformation Assay, Binding Assay, Activation Assay