Journal: Current biology : CB

Article Title: Microtubule-Dependent Confinement of a Cell Signaling and Actin Polymerization Control Module Regulates Polarized Cell Growth.

doi: 10.1016/j.cub.2018.05.076

Figure Lengend Snippet: Figure 4. Microtubules Confine SPK1 Signaling Nodules to the Branch Apex (A) YFP:SPK1 punctae are clustered within a microtubule-depletion zone. Right: representative intensity plot of YFP:SPK1 and mRUBY2:MBD signal along the cell periphery below the yellow line. (B) Microtubule organization in developing tri- chomes expressing GFP:MBD with and without oryzalin treatment. Microtubules are completely depolymerized after 1.5 hr of oryzalin treatment, but recovered after inhibitor washout. (C–E) SPK1 localization becomes progressively unfocused following oryzalin treatment, and the effect is reversible following inhibitor washout (C). Perimeter length of the SPK1 signal, normalized by tip radius of curvature, as a function of oryzalin treatment time and inhibitor washout (D). Signal intensity of the cortical SPK1 signal normalized to local cytosolic signal intensity as a function of oryzalin treatment time and inhibitor washout (E). Mean ± SD (n = 22). *p < 0.05, **p < 0.01 (ANOVA with Tukey HSD test). Scale bars represent 5 mm. See also Figure S4.

Article Snippet: The MYB5 promoter, mRUBY2 and MBD-nos terminator were amplified from pEGADMYB5pro (provided by D Marks, University of Minnesota, St. Paul, MN), pFA6a-link-yomRuby2-Kan (Addgene, Cambridge, MA) and GFP:MBD [36], respectively.

Techniques: Expressing

Journal: Nature protocols

Article Title: Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins

doi: 10.1038/s41596-018-0075-9

Figure Lengend Snippet: List of transfer plasmids.

Article Snippet: Proviral DNA sizes are noted in brackets. (D) Comparative size-exclusion chromatograms of secreted NET1 ΔNTR and NEO1 FN456 , showing improved secreted protein yields from lentiviral transduction (blue curves) as compared to transient transfection (red curves) of adherent HEK293T cells. table ft1 table-wrap mode="anchored" t5 caption a7 Plasmid no. pHR-CMV-TetO 2 Addgene Plasmid # Purpose 1 3C-Twin-Strep 113883 Protein (co-)expression, FACS 2 3C-Twin-Strep_IRES-EmGFP 113884 3 3C-Twin-Strep_IRES-mRuby2 113885 4 3C-Twin-Strep_IRES-mTurquoise2 113886 5 3C-Avi-His6 113887 Protein (co-)expression, FACS, in vivo biotinylation 6 3C-Avi-His6_IRES-EmGFP 113888 7 3C-Avi-His6_IRES-mRuby2 113889 8 3C-Avi-His6_IRES-mTurquoise2 113890 9 3C-mVenus-Twin-Strep 113891 Membrane protein expression and FSEC-TS screening, FACS 10 EmGFP 113892 Transduction & FACS compensation controls 11 mVenus 113893 12 mRuby2 113894 13 mTurquoise2 113895 14 HA-BirA 113896 HA-tagged cytosolic and ER-resident biotin ligase for in vivo biotinylation 15 HA-BirA-ER 113897 16 3C-Avi-His6_IRES-HA-BirA-ER 113898 Plasmid no. pHR-CMV Addgene Plasmid # Purpose 17 TetR-HA-NLS-P2A-BSD-Myc 113899 Generation of inducible cell lines Plasmid no. pHR-SFFV Addgene Plasmid # Purpose 18 3C-Twin-Strep 113900 Alternative for CMV-TetO 2 Plasmid no. pHR-CAG Addgene Plasmid # Purpose 19 3C-Twin-Strep 113901 Alternative for CMV-TetO 2 Open in a separate window caption a8 List of transfer plasmids.

Techniques: Plasmid Preparation, In Vivo, Expressing, Transduction