Journal: Human molecular genetics

Article Title: Altered pre-lamin A processing is a common mechanism leading to lipodystrophy.

doi: 10.1093/hmg/ddi158

Figure Lengend Snippet: Figure 1. Analysis of pre-lamin A expression and localization in control and laminopathic fibroblasts. (A) Western blot analysis of pre-lamin A in untreated skin fibroblasts. Whole cell lysates from control (C), MAD, FPLD, WS and EDMD2 fibroblasts were submitted to electrophoresis, western blotted and probed using anti-pre-lamin A antibody. Immunoblotted membranes were stripped and re-probed with anti-lamin A/C, anti-emerin or anti-actin antibodies (lower panels). Actin staining shows equal loading of samples. Molecular weight markers are reported in kilodalton. (B) Densitometric analysis of pre-lamin A immunoblotted bands shown in panel (A). Immunoblotted bands were quantified by densitometry. At least three independent experiments were performed for western immunoblot analysis of pre-lamin A accumulation, equal loading of samples was checked using actin as an internal loading control and data were calculated as percentage of control pre-lamin A densitometry obtained in each experiment. Values are reported as a percentage of control pre-lamin A amount (mean + SEM of three different experiments). Densitometric values obtained for untreated MAD, FPLD and WS samples are significantly different from control values, as calculated by Student’s t-test (P , 0.05). (C) Localization of pre-lamin A in control, MAD, FPLD, WS and EDMD2 fibroblasts. Double-staining of pre-lamin A and emerin was performed by anti-pre-lamin A polyclonal antibody (Santa Cruz, SC-6214, revealed by FITC-conjugated secondary antibody) and anti-emerin monoclonal antibody (revealed by Cy-3-conjugated secondary antibody). Pictures were obtained by fluorescence microscopy. Pre-lamin A-labeled intra-nuclear structures are marked by arrowheads.

Article Snippet: The antibodies employed for western blot analysis or immunofluorescence labeling were as follow: anti-lamin A/C, monoclonal (Novocastra Laboratories, NCL-LAM-A/C); anti-lamin A/C, goat polyclonal, (Santa Cruz, sc-6215); anti-pre-lamin A, goat polyclonal (Santa Cruz, sc-6214) (15); anti-SREBP1, rabbit polyclonal (Santa Cruz, sc-8984); anti-emerin, mouse monoclonal (Novocastra Laboratories, NCL-emerin); antiPPARg, mouse monoclonal (Santa Cruz, sc-7273); anti-actin, goat polyclonal (Santa Cruz, sc-1616) and anti-FLAG, rabbit polyclonal (Sigma, F-7425).

Techniques: Expressing, Control, Western Blot, Electrophoresis, Staining, Molecular Weight, Double Staining, Microscopy, Labeling

Journal: Human molecular genetics

Article Title: Altered pre-lamin A processing is a common mechanism leading to lipodystrophy.

doi: 10.1093/hmg/ddi158

Figure Lengend Snippet: Figure 2. Analysis of pre-lamin A expression and localization in mevinolin-treated control and laminopathic fibroblasts. (A) Western blot analysis of pre-lamin A in skin fibroblasts, following 18 h mevinolin treatment. Whole cell lysates from control (C), MAD, FPLD, WS and EDMD2 fibroblasts were submitted to electrophoresis, western blotted and probed using anti-pre-lamin A antibody. Immunoblotted membranes were stripped and re-probed with anti-lamin A/C, anti-emerin or anti-actin antibodies (lower panels). Actin staining shows equal loading of samples. Molecular weight markers are reported in kilodaltons. (B) Densitometric analysis of pre-lamin A immunoblotted bands shown in panel (A). Immunoblotted bands were quantified by densitometry. At least three inde- pendent experiments were performed for western immunoblot analysis of pre-lamin A accumulation. Equal loading of samples was checked using actin as an internal loading control and data were calculated as percentage of pre-lamin A densitometry measured in each untreated cell line. Values are reported as a per- centage of pre-lamin A amount in each corresponding untreated cell line (Fig. 1A and B) and they represent means + SEM of three different experiments. Western blotting of untreated fibroblasts (Fig. 1A) and mevinolin-treated fibroblasts (A) was routinely performed on the same membrane to allow comparison of results. (C) Localization of pre-lamin A in mevinolin-treated control, MAD, FPLD and WS fibroblasts. Double-staining of pre-lamin A and emerin was performed by anti-pre-lamin A polyclonal antibody (Santa Cruz, SC-6214, revealed by FITC-conjugated secondary antibody) and anti-emerin monoclonal anti- body (revealed by Cy-3-conjugated secondary antibody (C0) Localization of pre-lamin A in mevinolin-treated wild-type nuclei (control). Pictures were obtained at two different focal planes (lamina plane, equatorial plane). Pre-lamin A-labeled intra-nuclear structures shown in (C) and (C0) are marked by arrowheads. Pictures were obtained by fluorescence microscopy.

Article Snippet: The antibodies employed for western blot analysis or immunofluorescence labeling were as follow: anti-lamin A/C, monoclonal (Novocastra Laboratories, NCL-LAM-A/C); anti-lamin A/C, goat polyclonal, (Santa Cruz, sc-6215); anti-pre-lamin A, goat polyclonal (Santa Cruz, sc-6214) (15); anti-SREBP1, rabbit polyclonal (Santa Cruz, sc-8984); anti-emerin, mouse monoclonal (Novocastra Laboratories, NCL-emerin); antiPPARg, mouse monoclonal (Santa Cruz, sc-7273); anti-actin, goat polyclonal (Santa Cruz, sc-1616) and anti-FLAG, rabbit polyclonal (Sigma, F-7425).

Techniques: Expressing, Control, Western Blot, Electrophoresis, Staining, Molecular Weight, Membrane, Comparison, Double Staining, Labeling, Microscopy

Journal: Cells

Article Title: Autophagic Removal of Farnesylated Carboxy-Terminal Lamin Peptides

doi: 10.3390/cells7040033

Figure Lengend Snippet: Determination of the heterochromatin organization and cell proliferation activity. ( A ) Heterochromatin was stained with an anti-HP1β antibody. Representative images of HP1β staining in EGFP vector-, EGFP–NLS-59-R-CSIM-, and EGFP–NLS-PG-9-CSIM-transfected cells. Cytoplasmic HP1β staining shows the disorganized heterochromatin signals in EGFP–NLS-59-R-CSIM- and EGFP–NLS-PG-9-CSIM-transfected cells, as indicated by the arrowheads. Higher magnification images show the colocalization of DAPI and heterochromatin staining in the cytoplasm. Scale bar, 10 µm; ( B ) Ki67 was used to detect the actively replicating chromosomal DNA. Representative images of Ki67 staining and signals for EGFP fusion proteins are shown. Ki67 signals are barely detectable in EGFP–NLS-59-R-CSIM- and NLS-PG-9-CSIM-expressing cells, as indicated with circles; ( C ) Percentages of cells expressing both EGFP and Ki67. Nine hundred sixty cells expressing the EGFP vector, 919 cells expressing EGFP–NLS-59-R-CSIM, and 934 cells expressing EGFP–NLS-PG-9-CSIM were counted ( n = 3). Scale bar, 10 µm.

Article Snippet: After fixation, cells were subjected to indirect immunofluorescence staining with the following primary antibodies: goat-anti-lamin B1 (Santa Cruz Biotechnology, Heidelberg, Germany, M-20, sc-6217, 1:50), rabbit-anti-lamin C (Abcam, Cambridge, UK, ab125679, 1:500), mouse-anti-emerin (Leica Biosystems, NCL-Emerin, 4G5, 1:500), mouse-anti-NPC 414 (BioLegend, MMS-120P, Mab414, 1:2000), rabbit-anti-SUN1 (Sigma-Aldrich, HPA008346, 1:600), rabbit-anti-LC3B (Cell Signaling Technology #2775, 1:400), mouse-anti-LAMP-2 (Santa Cruz Biotechnology, sc-18822, 1:400), rabbit-anti-HP1β (Sigma-Aldrich, H2039,1:400), goat-anti-Ki67 (BD, 610968, 1:400), mouse-anti-EGFP (Clontech, 1:1000), rabbit-anti-progerin ( Monoclonal antibody [ ], 0.1 µg/mL), mouse-anti-progerin (Enzo, ALX-804-662, 13A4, 1:600), mouse-anti-Calnexin (Abcam, ab31290, 1:500), and mouse-anti-GM130 (BD, 610823, 1:100).

Techniques: Activity Assay, Staining, Plasmid Preparation, Transfection, Expressing

Journal: Cell reports

Article Title: Activity-dependent synthesis of Emerin gates neuronal plasticity by regulating proteostasis.

doi: 10.1016/j.celrep.2025.115439

Figure Lengend Snippet: Figure 4. Emerin bidirectionally regulates protein synthesis, including translation factors and synaptic proteins (A–C) BONCAT-western blot (BONCAT-WB) analysis of NSPs with Emerin KD and OE. (A) Experimental workflow for BONCAT and FUNCAT. DIV12 neuronal cultures were treated with AHA for 1 h, followed by BONCAT-WB or FUNCAT- immunolabeling. (B) BONCAT-WB biotin signal with Emerin KD (shEmd vs. control shScramble) (left) or OE (Emd-mRuby vs. control mRuby) in excitatory (center) and inhibitory neurons (right).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Emerin monoclonal primary antibody Cell signaling #30853S RRID: AB_2798996 Mouse anti-Lamin A/C monoclonal primary antibody Cell signaling #4777S RRID: AB_10545756 Goat anti-biotin polyclonal primary antibody Invitrogen #31852 RRID: AB_228243 Rabbit anti-cFos monoclonal primary antibody Cell signaling #2250S RRID: AB_2247211 Guinea pig anti-Map2 polyclonal primary antibody Synaptic System #188004 RRID: AB_2138181 Mouse anti-Map2 monoclonal primary antibody Sigma-Aldrich #M9942 RRID: AB_477256 Rabbit anti-PDI monoclonal primary antibody Cell signaling #3501 RRID: AB_2156433 Rabbit anti-Synaptophysin monoclonal primary antibody Synaptic System #101008 RRID: AB_2864779 Mouse anti-PSD95 monoclonal primary antibody Cell signaling #36233 RRID: AB_2721262 Rabbit anti-vGlut1 monoclonal primary antibody Synaptic System #135308 RRID: AB_2864787 Guinea pig anti-Homer1 polyclonal primary antibody Synaptic System #160004 RRID: AB_10549720 Rabbit anti-vGat polyclonal primary antibody Synaptic System #131013 RRID: AB_2189938 Mouse anti-Gephyrin monoclonal primary antibody Synaptic System #147021 RRID: AB_2232546 Bacterial and virus strains NEB Stable competent E. coli NEB Bio-Lab Cat#C3040H AAV.dj capsid Gift from Maximov lab / Chemicals, peptides, and recombinant proteins L-azidonorleucine Chem Impex Cat#30456 L-azidohomoalanine Fisher Scientific Cat#502107772 Bicuculline Methiodine Tocris Cat# 2503 Actinomycin-D Sigma Aldrich Cat#A1410 Anisomycin Tocris Cat#22862-76-6 Paraformaldehyde Electron Microscopy Sciences Cat#15710-S TBTA Sigma-Aldrich Cat#678937 CuSO4 Sigma-Aldrich Cat#451657 Biotin-PEG4-alkyne Click Chemistry Tools Cat#TA105-25 Heavy isotope-labeled-biotin-alkyne Setareh Cat#7889 Light isotope-labeled-biotin-alkyne Setareh Cat#7884 TCEP Sigma-Aldrich Cat#C4706 Critical commercial assays BCA protein assay ThermoFisher Cat#23225 Duolink proximity ligation assay Sigma Aldrich Duolink Western blot Bio-Rad MiniPROTEAN Deposited data VE-induced cell type-specific nascent proteomic analysis ProteomeXchange Accession # PXD050926 Emerin overexpression nascent proteomic analysis ProteomeXchange Accession # PXD050926 Experimental models: Organisms/strains EMX1Cre mice Jackson laboratory JAX 005628 vGatCre mice Jackson laboratory JAX 16962 R26lsl GFP-mMetRS mice Jackson laboratory JAX 028071 C57BL/6 mice Scripps Research breeding colony / (Continued on next page) Cell Reports 44, 115439, April 22, 2025 19

Techniques: Western Blot, Immunolabeling, Control

Journal: Cell reports

Article Title: Activity-dependent synthesis of Emerin gates neuronal plasticity by regulating proteostasis.

doi: 10.1016/j.celrep.2025.115439

Figure Lengend Snippet: Figure 6. Emerin is required for normal visual responses in mice (A–G) Visually-evoked potential (VEP) recordings with Emerin KD. (A) Experimental workflow of unilateral Emerin KD and bilateral VEPs recording. (B) Representative coronal brain image showing AAV targeting and coverage. Left hemispheres are marked during sectioning for post hoc data interpretation. (C) Workflow for pre-recording preparation. (D) Visual stimulation and data acquisition protocol. (E) Representative VEP traces when both eyes were covered (left) and with normal visual stimulation (right). Recordings from KD (blue) and control (gray) hemispheres are aligned to the stimulus. (F) Trough-peak amplitude comparing left vs. right hemispheres (left) and shEmd vs. shScramble treatments (right). (G) VEP latency comparing left vs. right hemispheres (left) and shEmd vs. shScramble treatments (right). n = 8 mice, paired t test. (H–J) VE-induced cFos expression with Emerin KD. (H) Experimental workflow of unilateral Emerin KD and VE paradigm. (I) Representative image of VE-induced cFos immunolabeling in the primary visual cortex. (J) cFos+ neurons in shEmd vs. shScramble treatments. n = 5 mice, paired t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Emerin monoclonal primary antibody Cell signaling #30853S RRID: AB_2798996 Mouse anti-Lamin A/C monoclonal primary antibody Cell signaling #4777S RRID: AB_10545756 Goat anti-biotin polyclonal primary antibody Invitrogen #31852 RRID: AB_228243 Rabbit anti-cFos monoclonal primary antibody Cell signaling #2250S RRID: AB_2247211 Guinea pig anti-Map2 polyclonal primary antibody Synaptic System #188004 RRID: AB_2138181 Mouse anti-Map2 monoclonal primary antibody Sigma-Aldrich #M9942 RRID: AB_477256 Rabbit anti-PDI monoclonal primary antibody Cell signaling #3501 RRID: AB_2156433 Rabbit anti-Synaptophysin monoclonal primary antibody Synaptic System #101008 RRID: AB_2864779 Mouse anti-PSD95 monoclonal primary antibody Cell signaling #36233 RRID: AB_2721262 Rabbit anti-vGlut1 monoclonal primary antibody Synaptic System #135308 RRID: AB_2864787 Guinea pig anti-Homer1 polyclonal primary antibody Synaptic System #160004 RRID: AB_10549720 Rabbit anti-vGat polyclonal primary antibody Synaptic System #131013 RRID: AB_2189938 Mouse anti-Gephyrin monoclonal primary antibody Synaptic System #147021 RRID: AB_2232546 Bacterial and virus strains NEB Stable competent E. coli NEB Bio-Lab Cat#C3040H AAV.dj capsid Gift from Maximov lab / Chemicals, peptides, and recombinant proteins L-azidonorleucine Chem Impex Cat#30456 L-azidohomoalanine Fisher Scientific Cat#502107772 Bicuculline Methiodine Tocris Cat# 2503 Actinomycin-D Sigma Aldrich Cat#A1410 Anisomycin Tocris Cat#22862-76-6 Paraformaldehyde Electron Microscopy Sciences Cat#15710-S TBTA Sigma-Aldrich Cat#678937 CuSO4 Sigma-Aldrich Cat#451657 Biotin-PEG4-alkyne Click Chemistry Tools Cat#TA105-25 Heavy isotope-labeled-biotin-alkyne Setareh Cat#7889 Light isotope-labeled-biotin-alkyne Setareh Cat#7884 TCEP Sigma-Aldrich Cat#C4706 Critical commercial assays BCA protein assay ThermoFisher Cat#23225 Duolink proximity ligation assay Sigma Aldrich Duolink Western blot Bio-Rad MiniPROTEAN Deposited data VE-induced cell type-specific nascent proteomic analysis ProteomeXchange Accession # PXD050926 Emerin overexpression nascent proteomic analysis ProteomeXchange Accession # PXD050926 Experimental models: Organisms/strains EMX1Cre mice Jackson laboratory JAX 005628 vGatCre mice Jackson laboratory JAX 16962 R26lsl GFP-mMetRS mice Jackson laboratory JAX 028071 C57BL/6 mice Scripps Research breeding colony / (Continued on next page) Cell Reports 44, 115439, April 22, 2025 19

Techniques: Control, Expressing, Immunolabeling

Journal: Cell reports

Article Title: Activity-dependent synthesis of Emerin gates neuronal plasticity by regulating proteostasis.

doi: 10.1016/j.celrep.2025.115439

Figure Lengend Snippet: Figure 7. Emerin is required for visual depth perception in mice (A) Side view schematic of the visual cliff arena (left) and top view showing the 25/75 distribution of safe and unsafe zones (right). (B) Experimental workflow of bilateral Emerin knockdown and visual cliff test sessions 5 days and 30 days after AAV injection. (C) Representative movement tracking of mice within the visual cliff arena using DeepLabCut. The safe zone is highlighted in gray. (D) Raster plots of time spent in safe (green) and unsafe (orange) zones of the shEmd and shScramble groups. (E–G) Quantification of behavioral parameters from the day 30 session. (E) Percent total time spent moving and relative time spent in the safe and unsafe zones. (F) Distance traveled in the entire arena, safe zone, and unsafe zone. (G) Average moving speed in the entire arena, safe zone, and unsafe zone. n = 6 mice, Welch’s t test between shEmd and shScramble. Lines indicate median and 95% CI. (H) Time spent in the safe zone comparing day 5 and day 30 after AAV injection. n = 6 mice, paired t test. Scale bars: 5 mm (B) and 200 mm (I).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Emerin monoclonal primary antibody Cell signaling #30853S RRID: AB_2798996 Mouse anti-Lamin A/C monoclonal primary antibody Cell signaling #4777S RRID: AB_10545756 Goat anti-biotin polyclonal primary antibody Invitrogen #31852 RRID: AB_228243 Rabbit anti-cFos monoclonal primary antibody Cell signaling #2250S RRID: AB_2247211 Guinea pig anti-Map2 polyclonal primary antibody Synaptic System #188004 RRID: AB_2138181 Mouse anti-Map2 monoclonal primary antibody Sigma-Aldrich #M9942 RRID: AB_477256 Rabbit anti-PDI monoclonal primary antibody Cell signaling #3501 RRID: AB_2156433 Rabbit anti-Synaptophysin monoclonal primary antibody Synaptic System #101008 RRID: AB_2864779 Mouse anti-PSD95 monoclonal primary antibody Cell signaling #36233 RRID: AB_2721262 Rabbit anti-vGlut1 monoclonal primary antibody Synaptic System #135308 RRID: AB_2864787 Guinea pig anti-Homer1 polyclonal primary antibody Synaptic System #160004 RRID: AB_10549720 Rabbit anti-vGat polyclonal primary antibody Synaptic System #131013 RRID: AB_2189938 Mouse anti-Gephyrin monoclonal primary antibody Synaptic System #147021 RRID: AB_2232546 Bacterial and virus strains NEB Stable competent E. coli NEB Bio-Lab Cat#C3040H AAV.dj capsid Gift from Maximov lab / Chemicals, peptides, and recombinant proteins L-azidonorleucine Chem Impex Cat#30456 L-azidohomoalanine Fisher Scientific Cat#502107772 Bicuculline Methiodine Tocris Cat# 2503 Actinomycin-D Sigma Aldrich Cat#A1410 Anisomycin Tocris Cat#22862-76-6 Paraformaldehyde Electron Microscopy Sciences Cat#15710-S TBTA Sigma-Aldrich Cat#678937 CuSO4 Sigma-Aldrich Cat#451657 Biotin-PEG4-alkyne Click Chemistry Tools Cat#TA105-25 Heavy isotope-labeled-biotin-alkyne Setareh Cat#7889 Light isotope-labeled-biotin-alkyne Setareh Cat#7884 TCEP Sigma-Aldrich Cat#C4706 Critical commercial assays BCA protein assay ThermoFisher Cat#23225 Duolink proximity ligation assay Sigma Aldrich Duolink Western blot Bio-Rad MiniPROTEAN Deposited data VE-induced cell type-specific nascent proteomic analysis ProteomeXchange Accession # PXD050926 Emerin overexpression nascent proteomic analysis ProteomeXchange Accession # PXD050926 Experimental models: Organisms/strains EMX1Cre mice Jackson laboratory JAX 005628 vGatCre mice Jackson laboratory JAX 16962 R26lsl GFP-mMetRS mice Jackson laboratory JAX 028071 C57BL/6 mice Scripps Research breeding colony / (Continued on next page) Cell Reports 44, 115439, April 22, 2025 19

Techniques: Knockdown, Injection