Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Schematic diagram showing interaction between the SETD2 C-terminal and HTT N-terminal proline-rich region (PRR), as reported in ( , ). ( B ) Immunoblot (IB) analysis showing coimmunoprecipitation of mCherry-tagged SETD2 C-terminal [SETD2-(C)-mCherry] and endogenous HTT in HEK293T cells. ( C ) IB analysis showing that actin methylation by SETD2 is dependent on both HTT and HIP1R using a SETD2 methyl-epitope antibody (anti-Me3 K36 ) to immunoprecipitate actin from 786-0 cells after siRNA-mediated knockdown of HTT or HIP1R . Input lysates shown to confirm knockdown of HTT and HIP1R. ( D and E ) IB analysis (D) and quantitation (E) of decreased insoluble F-actin in 786-0 cells after siRNA-mediated knockdown of HTT or HIP1R . Data are means ± SEM ( n = 3). ( F ) Quantitation of migration data after siRNA-mediated knockdown of HTT or HIP1R in SETD2-proficient versus SETD2-deficient 786-0 cells. Small circles each represent an independent measurement from all biological replicates. Large circles represent mean from 12 measurements for each independent biological replicate ( n = 4). ( G ) IB analysis showing decreased actin methylation after expression of a mutant HTT protein by IP of actin using a SETD2 methyl-epitope antibody (anti-Me3 K36 ) from HEK293T cells expressing a doxycycline (Doxy)–inducible CFP-tagged N-terminal HTT construct containing 94 polyglutamine repeats (CFP-HTT94Q) and a retrotransactivator (rtTA3). Input lysate confirms expression of CFP-tagged mutant protein and loss of SETD2 methylation activity (H3K36me3) without change in endogenous SETD2 expression. Data in (B), (C), and (G) are representative of experiments repeated three times with similar results.

Article Snippet: The following primary antibodies were used in this study: actin (C4, mouse; Santa Cruz Biotechnology, sc-47778), actin (C4, mouse; Millipore, MAB1501), actin (13E5, rabbit; Cell Signaling, 4970S), CFP (rabbit; Bio-Rad, AHP2986), GST (mouse; Santa Cruz Biotechnology, sc-138), HIP1R (rabbit; Proteintech, 16814-1-AP), histone H3 (D1H2, rabbit; Cell Signaling, 4620S), histone H3K36me3 (rabbit; Active Motif, 61101), HTT (1HU-4C8, mouse; Millipore, MAB2166), HTT (mouse; Bethyl, A302-812A), lamin A/C (rabbit; Cell Signaling, 2032S), lactate dehydrogenase (LDH; rabbit; Abcam, ab47010), mCherry (16D7, rat; Thermo Fisher Scientific, M11217), mCherry (rabbit; Abcam, ab167453), mCherry (mouse; Novus, NBP1-96752), pan–anti-trimethyllysine (rabbit; PTM Biolabs, PTM-601), SETD2 (rabbit; Abcam, ab31358), SETD2 (rabbit; ABclonal, A3194), SETD2 (rabbit; Invitrogen, PA5-83615), SETD2 (rabbit; Sigma-Aldrich, HPA-042451), SETD3 (rabbit; Abcam, ab176582), and tubulin (DM1A, mouse; Santa Cruz Biotechnology, sc-32293).

Techniques: Western Blot, Methylation, Knockdown, Quantitation Assay, Migration, Expressing, Mutagenesis, Construct, Activity Assay

Journal: Journal of Virology

Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein

doi: 10.1128/JVI.01910-18

Figure Lengend Snippet: Nuclear localization of Rep from two TYLCV strains is controlled by the conserved lysines. (A) Subcellular localization of RFP-tagged RepTYLCV (TYLCV-Alb13) variants in N. benthamiana upon transient expression with Agrobacterium. Arrowheads indicate fluorescence in the cytoplasm, and asterisks mark nonfluorescent or weakly fluorescent nuclei. Scale bars represent 5 μm. (B) Immunoblot of the Rep-RFP fusion proteins upon transient expression in N. benthamiana. Proteins were detected with an anti-RFP antibody. To demonstrate equal protein loading, the membranes were stained with Ponceau S. (C) Micrographs of epidermal leaf cells transiently expressing RepTYLCV K-to-A triple mutant-RFP and stained with ER-Tracker Green. From the left in order: RFP channel, ER-Tracker, merge of RFP and ER-Tracker, bright field, and graph representing the normalized fluorescence intensity of RFP and ER-Tracker (GFP) along the lines in the micrographs. Note that the two signals are shifted and do not overlap, indicating that they localize in different positions. Scale bars represent 50 μm. (D) Subcellular localization of RFP-tagged Rep TYLCV-Almeria WT and K-to-A variants in N. benthamiana. For each sample, one representative epidermal cell is shown with a 4× zoom of its nucleus; arrowheads indicate fluorescence in the cytoplasm, and asterisks mark nonfluorescent or weakly fluorescent nuclei. The scale bars represent 5 μm. (E) Box plot showing the RFP fluorescence intensity ratio in the cytoplasm versus nucleus for the images shown in panel D; a total of 16 cells were analyzed. (F) Box plot depicting the RFP fluorescence intensity ratio in the cytoplasm versus nucleus for the images shown in panel A; a total of 8 cells per sample were analyzed. The statistical analysis used is described in the legend to Fig. 2.

Article Snippet: Immunodetection of the proteins was performed according to standard protocols using anti-RFP antibody (Chromotek 6G6; 1:1,000) to detect the Rep-RFP fusion proteins and antihemagglutinin (anti-HA) antibody (Roche 3F10; 1:2,000) for Rep-SCFP C fusions as primary antibodies and anti-rat (Pierce 31470; 1:10,000) or anti-mouse (Pierce 31430; 1:10,000) as secondary antibodies.

Techniques: Expressing, Fluorescence, Western Blot, Staining, Mutagenesis