c las  (Bio-Rad)

Journal: PNAS Nexus

Article Title: Roles of the lamin A-specific tail region in the localization to sites of nuclear envelope rupture

doi: 10.1093/pnasnexus/pgae527

Figure Lengend Snippet: The localization kinetics of LA and PG at the rupture sites in WT and G609G/+ nuclei. A) Protein architecture of LA and PG. The coiled-coil central rod domain (gray), the NLS (yellow), the β-strand comprising the Ig-fold domain (blue), and the CaaX motif (orange) are shown in the diagram. Anti-LA (4A58) and anti-PG do not cross-react with each other. Anti-pre-LA (7G11) recognizes the last amino acids that is proteolytically cleaved off and does not react with PG. Anti-mature-LA (4A4-A4) recognizes mature-LA but not pre-LA. Anti-LA/C (3A6-4C11) recognizes all A-type lamin isoforms. B) NE rupture was induced by single-cell compression with a 100-μm bead attached on a cantilever, as shown in the diagram. C and D) Time-lapse images of NLS-sfCherry expressed in WT and G609G/+ cells were acquired with 30-s intervals for 5 min after the induction of NE rupture by single-cell compression. C) The fluorescence intensities in the cells are shown with rainbow color. Bar: 10 μm. D) The nuclear-to-cytoplasmic intensity (N/C) ratios of NLS-sfCherry were measured, normalized to the initial intensities, and plotted to monitor the nuclear reentry in the cells (means ± SEM; n = 20 cells from two independent experiments; ***, P < 0.001 from WT by a mixed-effects model). E) NE rupture was induced by 405-nm laser microirradiation with a 2-μm diameter spot at the NE of WT and G609G/+ nuclei. F and G) 13 to 21 of nuclei were laser-microirradiated within 10 min and incubated for 0, 30, 60, and 120 min, followed by fixation with 4% PFA/0.1% Triton-X 100. The fixed cells were immunostained with a combination of the anti-LA and anti-PG, and the DNA was stained with Hoechst 33342. F) Representative confocal images of nuclei in the cells fixed within 60–70 min after the induction of NE rupture by laser microirradiation. Magnified views of the areas indicated with orange boxes are shown (bottom). The rupture sites are indicated with yellow arrowheads (bottom). Bars: 5 μm (top) and 2μm (bottom). G) Percentiles of the cells with (blue) and without (gray) the localization of LA and PG to the rupture sites. The numbers of analyzed cells from two independent experiments are indicated in the bar charts. The line graphs show the kinetics of the percentage of the cells with localization of LA and PG to the rupture sites.

Article Snippet: Primary antibodies used for immunoblotting were mouse monoclonal anti-LA/C (1:5,000–20,000; 3A6-4C11, eBioscience), rat monoclonal anti-pre-LA (1:1,000; 7G11), rabbit polyclonal anti-PG (1:1,000; RaPG, Nourshargh lab), rabbit monoclonal anti-BANF1/BAF (1:500; EPR7668, Abcam), anti-β-actin (1:2,000; AC-15, Santa Cruz), and anti-GAPDH (1:5,000; 6C5, Santa Cruz).

Techniques: Fluorescence, Incubation, Staining

Journal: PNAS Nexus

Article Title: Roles of the lamin A-specific tail region in the localization to sites of nuclear envelope rupture

doi: 10.1093/pnasnexus/pgae527

Figure Lengend Snippet: Localization of LA and PG to the rupture sites in DMSO or FTI-treated WT and G609G/+ nuclei. A) Whole-cell lysates from WT and G609G/+ cells treated with 0.1% DMSO or 3.2 μM lonafarnib (FTI) were probed with anti-LA/C (3A6-4C11), anti-PG, and anti-GAPDH (as loading control) for immunoblotting. Positions of the size standards are shown on the right. B and C) The indicated cells were immunostained with the anti-LA (4A58) and anti-PG. B) Representative microscopic images of single confocal sections. Bar: 5 μm. C) Ratios of the average fluorescence intensity in the nucleoplasmic pool relative to that of the NL (NP/NL ratios) of LA and PG were measured based on immunofluorescence ( n = 20 cells from two independent experiments; ***, P < 0.001 by Welch's t tests). D) 15 to 19 of nuclei were laser-microirradiated and incubated for 60 min, followed by fixation with 4% PFA/0.1% Triton-X 100. The fixed cells were immunostained with the anti-LA and anti-PG. Percentiles of the cells with (blue) and without (gray) the localization of LA and PG at the rupture sites. The numbers of analyzed cells from two independent experiments are indicated in the bar charts.

Article Snippet: Primary antibodies used for immunoblotting were mouse monoclonal anti-LA/C (1:5,000–20,000; 3A6-4C11, eBioscience), rat monoclonal anti-pre-LA (1:1,000; 7G11), rabbit polyclonal anti-PG (1:1,000; RaPG, Nourshargh lab), rabbit monoclonal anti-BANF1/BAF (1:500; EPR7668, Abcam), anti-β-actin (1:2,000; AC-15, Santa Cruz), and anti-GAPDH (1:5,000; 6C5, Santa Cruz).

Techniques: Control, Western Blot, Fluorescence, Immunofluorescence, Incubation

Journal: PNAS Nexus

Article Title: Roles of the lamin A-specific tail region in the localization to sites of nuclear envelope rupture

doi: 10.1093/pnasnexus/pgae527

Figure Lengend Snippet: The localization of pre- and mature-LA in the control and Zmpste24-KD WT MEFs treated with DMSO or FTI. A) Whole-cell lysates from the control and Zmpste24-KD cells treated with DMSO or FTI were probed with anti-LA/C (3A6-4C11) and anti-GAPDH (as loading control). Positions of the size standards are shown on the right. B) The control and Zmpste24-KD cells treated with DMSO or FTI were immunostained with a combination of anti-pre-LA (7G11) and anti-mature-LA (4A4-A4). Bar: 5 μm. NP/NL ratios of pre-LA (C) and mature-LA (D) in the cells were measured based on immunofluorescence ( n = 20 cells from two independent experiments; ***, P < 0.001 by Games–Howell tests for pre-LA and by Welch's t test for mature-LA). E) 15 to 18 of nuclei were laser-microirradiated within 10 min and incubated for 60 min, followed by fixation with 4% PFA/0.1% Triton-X 100. The fixed cells were immunostained with a combination of the anti-pre-LA and anti-mature-LA. Percentiles of the indicated cells with (blue) and without (gray) the localization of LA and pre-LA to NE rupture sites. The numbers of analyzed cells from two independent experiments are indicated in the bar charts. C–D) N/A, not applicable due to below the detection limit.

Article Snippet: Primary antibodies used for immunoblotting were mouse monoclonal anti-LA/C (1:5,000–20,000; 3A6-4C11, eBioscience), rat monoclonal anti-pre-LA (1:1,000; 7G11), rabbit polyclonal anti-PG (1:1,000; RaPG, Nourshargh lab), rabbit monoclonal anti-BANF1/BAF (1:500; EPR7668, Abcam), anti-β-actin (1:2,000; AC-15, Santa Cruz), and anti-GAPDH (1:5,000; 6C5, Santa Cruz).

Techniques: Control, Immunofluorescence, Incubation