Journal: bioRxiv

Article Title: Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays

doi: 10.1101/343939

Figure Lengend Snippet: Agrobacterium-mediated leaf transformation with expression constructs was carried out followed by imaging three days later using 100X lens with confocal microscopy to detect DAPI (405 nm excitation laser), GFP (488 nm excitation laser), or mCherry/mRFP (561 nm excitation laser). Representative focal sections are shown for GFP (green), mCherry (green in panel A, bottom row) mRFP (majenta in panel B), or DAPI (majenta in panels A and D). (A) GFP-MLKG1, GFP-MLKP1, GFP-MLKP2 and mCherry-ZmSUN2 (green) are localized at the nuclear periphery, surrounding the nucleus (n) stained with DAPI (magenta). (B) GFP-MLKG1 (green) is also present in a network-like pattern at the cell periphery, where it partially co-localizes with the ER marker HDEL-mRFP (magenta, top row) and the actin marker lifeact-mRFP (magenta, bottom row). (C) Schematic overview depicting the terminal deletion constructs. (D) Images from deletion constructs (listed at left) are shown as GFP only (1st column), DAPI only (2nd column), or overlay (3rd column). GFP-MLKP1ΔKASH shows additional localization to the cytoplasm and the nucleoplasm (magenta), whereas the others retain a tendency to localize to the nuclear periphery. All scale bars 10μm.

Article Snippet: Coding sequences of MLKP1 and MLKP2 genes fused to the C-terminus of eGFP-FLAG-HA sequence were custom synthesized by Genscript as pHWBF08 and pHWBF06 respectively.

Techniques: Transformation Assay, Expressing, Construct, Imaging, Confocal Microscopy, Staining, Marker

Journal: bioRxiv

Article Title: Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays

doi: 10.1101/343939

Figure Lengend Snippet: GFP localization assays were performed as described for . (A) Schematic overview of domain composition of the MLKP1 constructs. (B) Staining patterns in and around the nucleus (1st three columns) and a the cell periphery (last column) shows that the construct lacking the TM domain and the GFP only are distributed throughout the cytoplasm and nucleoplasm. Scale bars for panel B are 5 μm. (C) Cell periphery images show the cytoplasm near the cell cortex for full length and domain deletion constructs. Gene names are listed on the left for each row; constructs are listed on the top for each type; n.t. = not tested). The MLKP1 terminal deletions (both Δ4 and ΔKASH) result in a more soluble staining pattern compared to the full length. The other deletions retain a pattern typical of ER association. Scale bars are 10 μm.

Article Snippet: Coding sequences of MLKP1 and MLKP2 genes fused to the C-terminus of eGFP-FLAG-HA sequence were custom synthesized by Genscript as pHWBF08 and pHWBF06 respectively.

Techniques: Construct, Staining

Journal: bioRxiv

Article Title: Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays

doi: 10.1101/343939

Figure Lengend Snippet: The fluorescent fusion protein (eGFP) constructs for MLKP1 and MKLP2 are depicted. (A) Schematic representation showing the location of the transmembrane and KASH domains in full length and deletion constructs. Three alanine residues added to the C-terminus of ΔKASH constructs are highlighted in yellow. (B) Pairwise protein sequence alignment (Clustal Omega) of MLKP1 and MLKP2 is shown. (C) Posterior probabilities (Phobius transmembrane topology predictor) of TM helix for C-termini of MLKP1 and MLKP2 are plotted.

Article Snippet: Coding sequences of MLKP1 and MLKP2 genes fused to the C-terminus of eGFP-FLAG-HA sequence were custom synthesized by Genscript as pHWBF08 and pHWBF06 respectively.

Techniques: Construct, Sequencing

Journal: bioRxiv

Article Title: Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays

doi: 10.1101/343939

Figure Lengend Snippet: Co-localization of GFP-MLKG1, GFP-MLKP1 and GFP-MLKP2 (green) with mCherry-ZmSUN2, mCherry-ZmSUN2ΔCC and mCherry-ZmSUN2ΔSUN (magenta) at the nuclear periphery. Size bar 5μm.

Article Snippet: Coding sequences of MLKP1 and MLKP2 genes fused to the C-terminus of eGFP-FLAG-HA sequence were custom synthesized by Genscript as pHWBF08 and pHWBF06 respectively.

Techniques:

Journal: bioRxiv

Article Title: Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays

doi: 10.1101/343939

Figure Lengend Snippet: (A) Schematic overview depicting the full length mCherry-ZmSUN2 and internal deletion constructs that removed the coiled coil domain (ΔCC) or the SUN domain (ΔSUN). Normalized averaged (n=26-37) intensity FRAP curves for (B) GFP-MLKG1, (D) GFP-MKLP1, or (F) GFP-MLKP2 expressed alone (green), with full-length mCherry-SUN2 (black), with mCherry-SUN2-ΔCC (blue), or with mCherry-SUN2-ΔSUN (pink). Plateau values are plotted to the right for assays with GFP-MLKG1 (C), GFP-MLKP1 (E), and GFP-MLKP2 (G). Box plot error bars represent SD (blue) and mean (red) values. ANOVA statistical analyses with multiple comparisons are noted (ns = not significant = p≥0.05, **** = significant with P ≤0.0001).

Article Snippet: Coding sequences of MLKP1 and MLKP2 genes fused to the C-terminus of eGFP-FLAG-HA sequence were custom synthesized by Genscript as pHWBF08 and pHWBF06 respectively.

Techniques: Construct

Journal: bioRxiv

Article Title: Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays

doi: 10.1101/343939

Figure Lengend Snippet: The recovery half times (t1/2(s)) are plotted for the full-length and co-expression experiments summarized in . The SD (blue lines) and mean (red lines) are shown for (A) GFP-MLKG1, (B) GFP-MLKP1, and (C) GFP-MLKP2. ANOVA statistical analyses with multiple comparisons are noted (ns = not significant = p≥0.05, ** = significant with P ≤0.01, **** = significant with P ≤0.0001). Replicate numbers range from n=26 to n=35, as listed in .

Article Snippet: Coding sequences of MLKP1 and MLKP2 genes fused to the C-terminus of eGFP-FLAG-HA sequence were custom synthesized by Genscript as pHWBF08 and pHWBF06 respectively.

Techniques: Expressing

Journal: bioRxiv

Article Title: Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays

doi: 10.1101/343939

Figure Lengend Snippet: Representative images of cells during the FRAP assays for the experiments summarized in . The FRAP ROIs (white boxes) are shown along with images taken at the times indicated across the top for (A) GFP-MLKG1, (B) GFP-MLKP1, and (C) GFP-MLKP2. Scale bar denotes 2μm. FRAP ROI shown by white box. All scale bars 2μm.

Article Snippet: Coding sequences of MLKP1 and MLKP2 genes fused to the C-terminus of eGFP-FLAG-HA sequence were custom synthesized by Genscript as pHWBF08 and pHWBF06 respectively.

Techniques:

Journal: International Journal of Alzheimer's Disease

Article Title: Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease

doi: 10.4061/2011/695413

Figure Lengend Snippet: SPTLC2 expression is reduced in the presence of functional AICD. (a) Mouse embryonic fibroblasts expressing an APP construct lacking the last 15 amino acids (aa) and therefore a functional AICD domain (MEF APP∆CT15) show increased SPT expression and activity compared to control fibroblasts (MEF WT). (b) MEF APP∆CT15 cells incubated with functional AICD peptide show compared to MEF APP∆CT15 cells incubated with solvent control decreased SPTLC2 expression. (c) MEF APP∆CT15 cells incubated with A β peptides and solvent control showed no difference in SPTLC2 expression. (d) SPTLC2 expression in Fe65 knock-down SH-SY5Y cells is increased.

Article Snippet: To determine the effect of A β 40 (10 ng/mL) and A β 42 (1 ng/mL) (B. Penke, Szeged, Hungary) or AICD (sequence in 1-letter code: KMQQNGYENPTYKFFEQMQN) (2 μ M) (Genscript Corporation, Piscatway, USA) synthetic peptides were incubated for 6 days in cell culture.

Techniques: Expressing, Functional Assay, Construct, Activity Assay, Control, Incubation, Solvent, Knockdown