Journal: bioRxiv

Article Title: Nucleoporin Elys attaches peripheral chromatin to the nuclear pores in interphase nuclei

doi: 10.1101/2023.08.16.553518

Figure Lengend Snippet: ( A , B , D ) Immunostaining of control, Lam-KD, Elys-KD, or (Lam-KD plus Elys-KD) S2 cells with anti-Elys, anti-Lam and Mab414 antibodies ( A ), with anti-Elys and Mab414 antibodies ( B ), or with anti-LBR, anti-Elys and anti-Lam antibodies ( D ). Scale bars 1 µm ( A , D ), 10 µm ( B ). ( C ) ImageJ quantification of Mab414 average fluorescence intensity (normalized on average Dapi fluorescence) across the NE in Elys-KD (two replicates, n = 75) and control (two replicates, n = 75) S2 cells. P value was estimated in a M-W U -test. ( E ) Western-blot analysis of proteins, co-immunoprecipitated with anti-Elys or anti-Nup107 antibodies from S2 extracts, probed by anti-Elys, or anti-Nup107 antibodies (n.i. – non-immune serum, IP/input ratio 1:4.5). ( F , G ) Immunostaining of S2 cells with anti-CenpA (kinetochores, violet), anti-Elys (red), anti-α-Tubulin (green) in metaphase ( F ), with anti-Elys (red), anti-Mab414 (green), anti-LBR (violet), Dapi (blue) in anaphase ( G ). Scale bars 1 µm ( F , G ). Arrows point to the Elys concentrated around decondensing chromatin ( G ). ( H ) Immunostaining of Elys-KD S2 cells with Mab414 antibodies (green) counterstained with Dapi (blue) in telophase. Scale bar 1 µm.

Article Snippet: As the primary, mouse monoclonal anti-Lam [ ] (ADL84, 1:500), guinea pig polyclonal anti-LBR [ ] (1:1000), mouse monoclonal Mab414 (1:300; Abcam ab24609), chicken polyclonal anti-CenpA [ ] (1:600), rabbit polyclonal α-Tubulin (1:2000; Abcam ab18251) and rabbit polyclonal anti-Elys [ ] (1:1000) antibodies were used.

Techniques: Immunostaining, Control, Fluorescence, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Epileptiform activity influences theta-burst induced LTP in the adult hippocampus: a role for lipid raft dynamics in early metaplasticity

doi: 10.1101/2022.12.06.519267

Figure Lengend Snippet: Each panel shows at the bottom the western-blot immunodetection of flotilin-1 ( A. ), caveolin-1 ( B. ), synaptophysin-1 ( C. ) PSD-95 ( D. ), gephyrin ( E. ), and alpha-tubulin ( F. ) obtained in one individual experiment where hippocampal slices underwent Schaffer collateral basal stimulation for 20 min and then were subjected either to interictal-like epileptiform activity (EA) induced by perfusion with 0Mg 2+ for 30 min or ictal-like EA by exposure to bicuculine (10 μM, Bic ) for 16 min, and then allowed to recover for 30 min before slice collection. Control slices were monitored for 70 min (the equivalent time of the EA protocol) before WB analysis. Western blot experiments were performed using synaptosome preparations obtained from these slices. Respective average change in total flotilin-1 ( A. ), caveolin-1 ( B. ), synaptophysin-1 ( C. ) PSD-95 ( D. ), gephyrin ( E. ), and alpha-tubulin ( F. ) immunoreactivities are also plotted at the top in each panel. Individual values and the mean ± S.E.M of five independent experiments are depicted. 100% - averaged PSD-95, gephyrin, caveolin-1, flotillin-1, or synaptophysin-1 immunoreactivity in control conditions (CTR, absence of EA). * P < 0.05 (ANOVA, Sidak’s multiple comparison test) as compared to CTR; ns represents non -significant differences P > 0.05 ( ANOVA , Sidak’s multiple comparison test) between respective bars.

Article Snippet: Membranes were incubated overnight at 4oC with mouse anti-gephyrin (#147011, Synaptic Systems, AB_2810214), rabbit anti-PSD-95 (#CST-2507, Cell Signalling Tech., AB_561221), mouse anti-caveolin-1 (#ab106642, Abcam, AB_10861399), mouse anti-flotillin-1 (#ab133497, Abcam, AB _11156367), rabbit antiphospho-Ser845-GluA1 (1:2500, Abcam #Ab76321; RRID: AB_1523688), rabbit antiphospho-Ser-831-GluA1 (1:2000, Abcam #Ab109464; RRID: AB_10862154), rabbit anti-GluA1 (1:4000, Millipore # AB1504; RRID:AB_2113602), rabbit anti-GluA2 (1:1000, Proteintech #11994-1-AP; RRID: AB_2113725) and rabbit anti-alpha-tubulin (1:5000, Proteintech #11224-1-AP; RRID: AB_2210206) or rabbit anti-beta-actin (1:10000, Proteintech, Cat# 60008-1; RRID:AB_2289225) primary antibodies.

Techniques: Western Blot, Immunodetection, Activity Assay, Control, Comparison

Journal: bioRxiv

Article Title: HDAC6 promotes self-renewal and migration/invasion of rhabdomyosarcoma

doi: 10.1101/823864

Figure Lengend Snippet: (A) Western blots against acetylated alpha-Tubulin and alpha-Tubulin in RD and Rh 5 cells with targeted disruption of HDAC6 by CRISPR or treated with DMSO (vehicle) and Tubastatin A (200 nM). (B) Scratch assay assessing effects of adding back wild-type (wt) HDAC6 and catalytically-dead (cd) HDAC6 in RD cells with HDAC6 knockout. Results are shown from one representative experiment of at least 3 repeats. (C) Phalloidin staining in RD cells with safe-harbor control region and HDAC6 CRISPR targeting following serum starvation and 15-minute EGF (50 ng/mL) treatment. Arrowheads point to representative areas of membrane ruffles and filopodia formation. Green = phalloidin, Blue = DAPI. (D) Double IF against HDAC6 (green) and RAC1 (red) in RD cells. (E) RAC1 GTP pulldown assay in RD cells harboring safe-harbor control and HDAC6 CRISPR targeting. (F) Summary of scratch assays assessing the effects of overexpressing GFP as a control, RAC1V12, RAC1N17 and RHOAV14 in the presence of CRISPR-mediated targeted disruption of HDAC6 in RD cells following 24 hours of serum starvation and 15 minutes of EGF (50 ng/mL) treatment. (G) Summary of cell growth change by cell counts over 6 days assessing the effects of overexpressing GFP as a control, RAC1V12 and RAC1N17 on cell growth of RD cells harboring targeted disruption of HDAC6 . Results for the average of 4 replicates for each condition in one of 3 independent experiments are shown. (H) RAC1-GTP pulldown assay in RD cells overexpressing GFP, RAC1V12 and RAC1N17 following 24 hours of serum starvation and 15 minutes of EGF (50 ng/mL) treatment. Each error bar in B, F and G represents standard deviation. NS = no significance, p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: The following antibodies including dilutions were used: rabbit polyclonal anti human mouse monoclonal anti human Ki- 67, (1:100, clone: MIB1, Dako), rabbit monoclonal anti human HDAC6 (1:100, clone, D2E5; Cell Signaling), mouse monoclonal anti human RAC1 (1:100, clone 102; BD Biosciences), mouse monoclonal anti acetylated alpha-tubulin (1:200; clone 6-11B-1; Santa Cruz Biotechnology) and mouse monocloncal anti-alpha-tubulin (1:200; B-5-1-2; Santa Cruz Biotechnology).

Techniques: Western Blot, CRISPR, Wound Healing Assay, Knock-Out, Staining, Standard Deviation