rabbit anti-lemd2  (Abnova)

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative Western blot of the indicated NE proteins in WT(HA-CSA) and CS-A cells. (B) Representative immunofluorescence images of lamin A/C, lamin B1, emerin, and SUN1 staining; scale bar: 25 μm. (C) Representative immunofluorescence staining showing total and insoluble LEMD2 protein in WT(HA-CSA) and CS-A cells; scale bar: 25 μm. (D) Quantification of the insoluble LEMD2 showing a significant reduction in CS-A compared with WT(HA-CSA) using a two-tailed unpaired t test (** P < 0.01), n = 3 with >100 cells per experiment. (E) Representative immunoblot showing LEMD2 expression level in the soluble, insoluble, and whole cell extracts in WT(HA-CSA) and CS-A cells. (E, F) Quantification of the relative LEMD2 expression level from the western blots as shown in (E); normalized to GAPDH, histone H3, and α-tubulin, respectively. P -values were calculated using a two-tailed paired t tests (ns P > 0.05, * P < 0.05). All experiments in this figure were n = 3 independent experiments. Source data are available for this figure.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Western Blot, Immunofluorescence, Staining, Two Tailed Test, Expressing

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: Representative immunofluorescence images of LEMD2 in pre-extracted WT(HA-CSA) and CS-A cells following siRNA-mediated knockdown of LEMD2 (siLEMD2); scale bar: 50 μm.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunofluorescence, Knockdown

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative immunofluorescence staining showing insoluble LEMD2 protein in WT AG10803 and CSA KO AG10803 cells; scale bar: 25 μm. (C) Quantification of the insoluble LEMD2 showing a significant reduction in CSA KO AG10803 compared with WT AG10803 cells using a two-tailed unpaired t test (** P < 0.01), n = 3 with >100 cells per experiment. (B) Representative immunofluorescence staining showing the appearance of F-actin stress fibers in CS-A cells compared with WT(HA-CSA), scale bar: 25 μm.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative confocal images of DAPI and PLA signal in the indicated cells using either anti-lamin A/C or anti-LEMD2 antibodies alone (negative controls) or in combination; scale bar: 50 μm. (B) Quantification of the number of PLA foci per nuclei. P -value was calculated using a one-way ANOVA test followed by Tukey’s post hoc test (**** P < 0.0001), n = 3 with >100 cells per experiment. (C) Representative immunofluorescence staining of DAPI and lamin B1 in WT(HA-CSA) and CS-A cells transfected with GFP and LEMD2-GFP-containing constructs. (D, E) Quantification of the nuclear form factor and (E) percentage of nuclear blebs in GFP expressing cells, n = 3 with >100 cells per experiment. P -value was calculated using a one-way ANOVA test followed by Tukey’s post hoc test (* P < 0.05, *** P < 0.001, **** P < 0.0001). All experiments in this figure were n = 3 independent experiments.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Transfection, Construct, Expressing

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Immunoprecipitation of LEMD2-GFP in WT(HA-CSA) cells overexpressing GFP+Flag-CSA (control) and Flag-CSA+LEMD2-GFP. (B) Representative time-lapse confocal images showing pre-bleach, bleach, and post-bleach images of the WT(HA-CSA) and CS-A nuclei with LEMD2-GFP overexpression. The red box indicates the photobleached region at the nuclear periphery. (C) Graph showing the FRAP kinetics of LEMD2-GFP expressed in WT(HA-CSA) (n = 25 cells) and CS-A (n = 25 cells) cells. Error bars represent SD. Scale bar: 5 μm. (D) Summary table showing the mean percentage of the immobile fraction and the mean half-time of recovery of LEMD2-GFP FRAP experiment. Data were compared using a two-tailed unpaired t test. All experiments in this figure were n = 3 independent experiments. Source data are available for this figure.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunoprecipitation, Control, Over Expression, Two Tailed Test

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: Details of primary and secondary antibodies used for immunofluorescence and Western blot experiments.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunofluorescence, Western Blot

Journal: Molecular Biology of the Cell

Article Title: BAF facilitates interphase nuclear membrane repair through recruitment of nuclear transmembrane proteins

doi: 10.1091/mbc.E20-01-0009

Figure Lengend Snippet: BAF recruits lamin A/C, emerin, and LEMD2 to promote nucleus recompartmentalization. (A) Representative fixed images of U2OS RFP-NLS shLMNB1 cells transfected with siControl (siCtl) or siBAF undergoing nuclear membrane rupture, determined by cytoplasmic RFP-NLS. Cells were labeled with antibodies to lamin A/C, emerin, or LEMD2. RFP-NLS images are gamma adjusted. Arrowheads, estimated rupture site. Scale bars = 10 μm. (B) Proportion of rupture sites with accumulated lamin A/C ( n values: siCtl, 50; siBAF, 55), emerin (siCtl, 62; siBAF, 70), or LEMD2 (siCtl, 92; siBAF, 95). N values are pooled from three experiments. *** p < 0.001, Fisher’s exact test. (C) Quantification of nucleus rupture durations after transfection with siRNAs against LMNA, EMD, or LEMD2. Data for siCtl and siBAF collected at the same time are reproduced from (open bars) ( n values: siLMNA, 548; siEmerin, 416; siLEMD2, 887 ruptures from three experiments). *** p < 0.0001, K-W test. (D) Histogram of proportion of ruptures shown in (C) with indicated durations of nucleus rupture. *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05, Fisher’s exact test.

Article Snippet: Rabbit anti–Lamin A/C (1:1,000; clone EPR4100; ab108595; Abcam) Mouse anti–Lamin B1 (1:100; clone C12; sc-365214; Santa Cruz Biotechnology) Mouse anti–Emerin (1:100; clone 4G5; EMERIN-CE; Leica Biosystems) Rabbit anti–LEMD2 (1:100; HPA017340; Sigma-Aldrich) Rabbit anti–cGAS (1:100; clone D1D3G; 15102S; Cell Signaling Technology)

Techniques: Transfection, Labeling