Journal: bioRxiv

Article Title: Hypothalamic extraretinal photoreceptor Opsin3 regulates water balance, body temperature and motor activity

doi: 10.1101/2022.07.28.501815

Figure Lengend Snippet: Arginine vasopressin ( Avp ), Caprin family member 2 ( Caprin2) , Cocaine and Amphetamine Regulated Transcript ( Cartpt) , Cellular retinoic acid-binding protein 1 ( Crabp1 ), Galanin ( Gal ), glucagon-like peptide-1 receptor ( Glp1r ), 5-Hydroxytryptamine Receptor 2A ( Htr2a ), oxytocin ( Oxt ), Prodynorphin ( Pdyn ), Pro-Melanin Concentrating Hormone ( Pmch ), ras-related dexamethasone induced 1 ( Rasd1 ), and Transthyretin ( Ttr ) were corrected for Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) RNA content. Data are expressed as mean ± SD.

Article Snippet: Following this, sections were incubated overnight at 4°C with primary antibodies against AVP neurophysin II (NP-II; MERCK, MABN845, PS41; 1:200); OXT NP-I (PS38; 1:100; ; CAPRIN2 (Proteintech, 20766-1-AP, 1:500), PDYN (Proenkephalin B (D-6); Santa Cruz Biotechnology, sc-398808, 1:100); CART (Bio-techne, AF163, 1:200); TTR (Bio-techne, NBP2-41101, 1:200), 5-HT2A (Alomone labs, ASR-033, 1:200).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Hypothalamic extraretinal photoreceptor Opsin3 regulates water balance, body temperature and motor activity

doi: 10.1101/2022.07.28.501815

Figure Lengend Snippet: Detection of green fluorescent protein (GFP), arginine vasopressin (AVP), oxytocin (OXT), Caprin family member 2 (CAPRIN2), Prodynorphin (PDYN), Cocaine- and amphetamine-regulated transcript protein (CART), Transthyretin (TTR) and Serotonin 5-HT2A receptor (5-HT2A). Images are representative of n = 4. Scale bar represents 40 µm.

Article Snippet: Following this, sections were incubated overnight at 4°C with primary antibodies against AVP neurophysin II (NP-II; MERCK, MABN845, PS41; 1:200); OXT NP-I (PS38; 1:100; ; CAPRIN2 (Proteintech, 20766-1-AP, 1:500), PDYN (Proenkephalin B (D-6); Santa Cruz Biotechnology, sc-398808, 1:100); CART (Bio-techne, AF163, 1:200); TTR (Bio-techne, NBP2-41101, 1:200), 5-HT2A (Alomone labs, ASR-033, 1:200).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation *

doi: 10.1074/jbc.M116.744607

Figure Lengend Snippet: Caprin-2 promotes constitutive LRP6 Ser-1490 phosphorylation. A, HEK293 cells were transfected with Caprin-2-HA. Cells were treated with control conditioned medium (CON CM), Wnt-3a protein, or Dkk1 CM for 30 min, and phosphorylation of endogenous LRP6 was then detected using an anti-pLRP6 (p1490) antibody. The anti-LRP6 antibody and anti-α-tubulin were used as the internal controls. The immunoblots were quantified by densitometry, and the intensity values were normalized with those of LRP6; values are given beneath each band. B, HEK293 cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 72 h. Cells were treated with control CM, Wnt-3a protein, or Dkk1 CM for 30 min, and phosphorylation of LRP6 was then detected and quantified as indicated. C, HEK293 cells were transfected with CDK14-GFP, Cyclin Y-GFP, and Caprin-2-HA, and 24 h later, cells were collected and analyzed by Western blotting. D, HEK293 cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 48 h, and then cells were transfected with CDK14-GFP and Cyclin Y-GFP. 24 h later, cells were collected and analyzed by Western blotting.

Article Snippet: Caprin-2 antibody was produced by Abgent.

Techniques: Transfection, Western Blot, Infection

Journal: The Journal of Biological Chemistry

Article Title: Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation *

doi: 10.1074/jbc.M116.744607

Figure Lengend Snippet: Caprin-2-mediated LRP6 Ser-1490 phosphorylation is cell cycle-dependent. A, HeLa cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 56 h. Then cells were arrested in G2/M by nocodazole (100 ng/ml) treatment for 16 h. Cells were collected in SDS sample buffer and analyzed by Western blotting. Parallel FACS analysis was performed correspondingly. B, HeLa cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 24 h. Then cells were synchronized at G1/S by double thymidine block. G1/S-arrested cells were then washed to progress through the cell cycle. Cells were collected at the indicated times and analyzed by Western blotting. FACS analysis of the cells is shown beneath the corresponding lanes. CCNB was used as a marker for cell cycle progression. C, HeLa cells with or without infection of Caprin-2 shRNAs were arrested at the indicated phases of the cell cycle by nocodazole treatment and then subjected to Western blotting analysis.

Article Snippet: Caprin-2 antibody was produced by Abgent.

Techniques: Infection, Western Blot, Blocking Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation *

doi: 10.1074/jbc.M116.744607

Figure Lengend Snippet: Caprin-2 interacts with CDK14/Cyclin Y. A, HeLa cells were fractionated and then subjected to Western blotting analysis. LRP6, α-tubulin, and Sp1 served as the loading controls for the membrane (Mem), cytosolic (Cyto), and nuclear (Nuc) fractions, respectively. B, schematic of Caprin-2 fragments. Numbers indicate amino acids. HR, homologous region. N, amino-terminal fragment of Caprin-2; M, middle fragment of Caprin-2; C, carboxyl-terminal fragment of Caprin-2. C and D, HEK293T cells expressing HA-Ca2-FL, HA-Ca2-N, HA-Ca2-M, or HA-Ca2-C were co-transfected with either CDK14-GFP or Cyclin Y-GFP. Then the cell lysates were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were probed with the indicated antibodies. E and F, GST-tagged CDK14 or Cyclin Y expressed in E. coli was bound to the glutathione-agarose beads. Then the His-tagged Caprin-2 fragments were added as indicated and incubated for 3 h. After three washes with lysis buffer, the proteins were eluted with the SDS loading buffer and analyzed by Western blotting using anti-GST and anti-His monoclonal antibodies.

Article Snippet: Caprin-2 antibody was produced by Abgent.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Incubation, Lysis

Journal: The Journal of Biological Chemistry

Article Title: Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation *

doi: 10.1074/jbc.M116.744607

Figure Lengend Snippet: Caprin-2 scaffolds the CDK14-Cyclin Y-LRP6 complex. A, HEK293T cells were transfected with CDK14-GFP and Cyclin Y-FLAG with or without 5HA-Caprin-2 as indicated. Cell lysates were then immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates were probed with the indicated antibodies. B, HEK-293T cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 48 h, and then cells were transfected with the indicated plasmids. Co-IP was performed as described in A. C, endogenous interaction of Caprin-2/CDK14/Cyclin Y increased at G2/M. HeLa cells were synchronized by double thymidine block, and the cells lysates from G1/S- or G2/M-synchronized cells were immunoprecipitated by Cyclin Y-specific antibody, followed by Western blotting analysis with the indicated antibodies. D, endogenous Caprin-2/CDK14/Cyclin Y/LRP6 complexes were analyzed in HEK293 cells. Immunoprecipitation was performed with an anti-LRP6 polyclonal antibody. IgG was used as a control. Before co-immunoprecipitation, cells were treated with shCaprin-2 lentivirus for 56 h and nocodazole for another 16 h. E, model for Caprin-2 modulating constitutive LRP6 phosphorylation.

Article Snippet: Caprin-2 antibody was produced by Abgent.

Techniques: Transfection, Immunoprecipitation, Infection, Co-Immunoprecipitation Assay, Blocking Assay, Western Blot