Rabbit anti lamin b1

The primary antibodies used in immunofluorescence were rabbit anti‐γ‐H2Ax (Cell Signalling), mouse anti‐cGAS (Santa Cruz Biotechnology), rabbit anti‐lamin B1(Cell Signalling), rabbit anti‐NFkB (p65) (Cell Signalling) while the secondary were Cy3 or FITC–conjugated goat anti‐rabbit or mouse IgG (Sigma‐Aldrich Chemicals). DAPI were used for nucleus staining. The coverslips with the immune‐labelled cells were mounted with an anti‐fade mounting medium (Biomeda, Collegno, Italy) and analysed under a Bio‐Rad MRC 1024 ES confocal laser scanning microscope (Bio‐Rad) equipped with a 15‐mW Krypton/Argon laser source. The cells were observed with a Nikon Plan Apo X60 oil immersion objective (Nikon Instruments, Rome, Italy) at 595 nm. Series of optical sections (X‐ and Y‐steps: 512 × 512 pixels) were then obtained through the depth of the cells, with a thickness of 1 μm at intervals of 0.8 μm (Z‐step). A single composite image was obtained by superimposition of 20 optical sections for each sample. Total NFkB fluorescence intensity and Mander's coefficient (M1), used to assess NFκB p65 colocalization with the nucleus (DAPI), were determined by ImageJ software.

Thirty‐forty micrograms of lysate proteins for each sample together with the molecular weight Magic Mark (Invitrogen, Carlsbad, CA, USA) were subjected to 4–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis separation (Bis‐Tris Plus BOLT, Invitrogen) and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Burlington, MA, USA). The membranes were blocked in 5% skim milk and incubated overnight with the following specific primary antibodies: rabbit anti‐lamin B1 (Cell Signalling), mouse anti‐Tubulin (Sigma) followed by the suitable HRP‐conjugated secondary antibodies (Sigma‐Aldrich Chemicals). All resulting immunocomplexes were visualized with an enhanced chemiluminescence ECL detection system (GE Healthcare, Milano, Italy) and quantified by ImageJ software (NIH, Bethesda, MD, USA).

The primary antibodies used in this study were rabbit anti-cleaved caspase 3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin (1:3000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-NF-κB p65 (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Lamin B1 (1:1000, Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-TXNDC9 (1:200, Abcam, Cambridge, UK). Species-specific secondary antibodies were purchased from Abcam (Cambridge, UK).

Nuclear extracts were prepared as previously described (Maestro et al. 2003 (link)) and separated on a 7% SDS-PAGE gel and transferred to an Immobilon polyvinylidene difluoride membrane (Millipore). Immunodetection was performed with mouse 12C11 anti-REST (1:1000) or rabbit anti-LaminB1 (1:2000; Cell Signaling). Quantification was performed with ImageJ-Fiji.