Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: SSeCKS sequesters cyclin D1 in glomerular parietal epithelial cells and influences proliferative injury in the glomerulus.

doi: 10.1038/labinvest.2011.199

Figure Lengend Snippet: Figure 2 Expression and interaction of Src-suppressed protein kinase C substrate (SSeCKS) and cyclin D1 in cultured parietal epithelial cells (PECs). (a) Fluorescence detection of SSeCKS (green), cyclin D1 (red), and nuclei (blue) during increasing cell–cell contact between PECs from 50 to 100% cell confluency showing induction of SSeCKS in the cytoplasm with concomitant nuclear to cytoplasmic redistribution of cyclin D1, compartmentalization that remains in fully differentiated (FD) PECs. (b) Western immunoblots (IB) showing downregulation of cyclin D1 and its immunoprecipitation (IP) with cytoplasmic SSeCKS during increasing cell–cell contact between PECs from 50 to 100% cell confluency, binding that remains in fully differentiated PECs.

Article Snippet: For immunoprecipitation of cyclin D1 with SSeCKS, 100 mg of protein from capsulated glomeruli or from cultured, differentiated PECs was incubated with rabbit polyclonal anti-SSeCKS antibody overnight at 41C, followed by binding to protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for SDS-PAGE separation of cyclin D1 and SSeCKS.

Techniques: Expressing, Cell Culture, Fluorescence, Western Blot, Immunoprecipitation, Binding Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: SSeCKS sequesters cyclin D1 in glomerular parietal epithelial cells and influences proliferative injury in the glomerulus.

doi: 10.1038/labinvest.2011.199

Figure Lengend Snippet: Figure 3 Phosphorylation of Src-suppressed protein kinase C substrate (SSeCKS) by activated protein kinase C (aPKC) in cultured parietal epithelial cells (PECs). (a) Western immunoblots showing rapid phosphorylation of SSeCKS at serine 283 (phospho-SSeCKS ser283) within 5 min of activation of PKC in PECs by phorbol 12,13-diacetate (PDA) or tumor necrosis factor-a (TNF-a), signaling abrogated by staurosporine aglycone (inhibitor). (b) Control western immunoblots showing the same signaling as in panel a in mouse fibroblasts expressing SSeCKS. (c) Fluorescence detection of phospho-SSeCKS ser283 (green), cyclin D1 (red), and nuclei (blue) in fully differentiated PECs showing phosphorylation of SSeCKs and cytoplasmic-to-nuclear redistribution of cyclin D1 following activation of PKC.

Article Snippet: For immunoprecipitation of cyclin D1 with SSeCKS, 100 mg of protein from capsulated glomeruli or from cultured, differentiated PECs was incubated with rabbit polyclonal anti-SSeCKS antibody overnight at 41C, followed by binding to protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for SDS-PAGE separation of cyclin D1 and SSeCKS.

Techniques: Phospho-proteomics, Cell Culture, Western Blot, Activation Assay, Control, Expressing, Fluorescence

Journal: Neuron

Article Title: Regulation of Thalamic and Cortical Network Synchrony by Scn8a

doi: 10.1016/j.neuron.2017.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were incubated in secondary antibodies: biotinylated anti-rabbit IgG (1:300, Vector Laboratories) and fluorescein avidin D (1:300, Vector Laboratories), or Alexa Fluor 555 anti-mouse IgG (1:1000, Thermo Fisher) or AlexaFluor 488 goat anti-mouse (1:1000, Thermo Fisher).

Techniques: Plasmid Preparation, Recombinant, Avidin-Biotin Assay, Software, Imaging

Journal: The Journal of Biological Chemistry

Article Title: Cellular requirements for iron–sulfur cluster insertion into the antiviral radical SAM protein viperin

doi: 10.1074/jbc.M117.780122

Figure Lengend Snippet: Model for the mode of interaction of CIA-targeting factors with viperin and for the unique maturation pathway. A, multimeric CIA-targeting complexes are present in the cell. As shown here, the CIA1–CIA2B–MMS19 complex interacts with the C terminus of viperin via direct contacts of both CIA1 and CIA2B. The two factors in turn recruit MMS19 to viperin. CIA2A binds at the N terminus of viperin and is stabilized by CIA1. Insertion of the radical SAM Fe/S cluster in the middle domain is mediated by C-terminally bound CIA1 only (see Ref. 6 and this study). B, Fe/S cluster maturation of viperin represents a novel branch of the CIA pathway that is initiated by the mitochondrial ISC machinery, early-acting CIA components, and IOP1 (20, 21). The components of the CIA1–CIA2B–MMS19-targeting complex operate in various combinations to facilitate Fe/S cluster insertion into dedicated client proteins, including GPAT, POLD1, and DPYD. CIA2A is specifically required for the maturation of IRP1 thus affecting cellular iron homeostasis and is stabilized by binding to CIA1. In contrast to all other known client Fe/S proteins, CIA1 appears to mediate Fe/S cluster insertion into the radical SAM protein viperin independently of the other CIA-targeting factors, thus representing a minimal requirement for CIA proteins.

Article Snippet: The rb α-FLAG epitope (F7425, d 1:5000), ms α-FLAG epitope (200472, d 1:2500), and rb α-POLD1 (15646–1-AP, d 1:1000) were obtained from Sigma, Stratagene, and Proteintech, respectively.

Techniques: Binding Assay