Western Blot Protocol for Protein Analysis

Cells were grown in 6-well plates (Greiner Bio-One, 657102) or 12-well plates (Thermo Scientific, 150628) and lysed using M-PER® Mammalian Protein Extraction Reagent (Thermo scientific, 78503). Protein concentration was measured with the Pierce™ BCA Protein Assay Kit (Thermo scientific, 23227). Cell lysates were mixed with 25% NuPage LDS sample buffer (ThermoFisher, NP0007) and 5% dithiothreitol (DDT, ThermoFisher NP0009) and heated for 10 min on 70 °C. Samples (5 µg) were loaded onto NuPAGE™ Novex 4–12% Bis–Tris Protein Gels (ThermoFisher, NP0322PK2), with MOPS running buffer (Thermo Scientific, J00047). PageRuler P-prestained Protein Ladder was used as marker (ThermoFisher, PI26616). Next, proteins were transferred to BioTrace PVDF membranes (Pall Corporation, 66542) using a transfer mixture of NuPAGE transfer buffer (ThermoFisher), NuPAGE antioxidant (ThermoFisher) and methanol. Afterwards the membranes were blocked in blocking buffer (5% ECL (Sigma GERPN418) in Tris Buffered Saline with 0.2% Tween 20 (TBST)), and subsequently incubated with primary antibodies, diluted in blocking buffer. The following primary antibodies were used: mouse anti-lamin A/C (Santa Cruz Biotechnology, sc-376248, 1/100), rabbit anti-lamin B1 (Abcam, ab16048, 1/1000), rabbit anti-lamin B2 (Abcam ab151735, 1/1000), mouse anti-H3K9me2,3 (Cell Signaling Technology, #5327, 1/1000), rabbit anti-H3K9ac (Cell Signaling Technology, #9649, 1/1000) and anti-cGAS (Cell Signaling Technology, 15102S, 1/1000). Rabbit anti-Nucleolin (Novus Biologicals, NB600-241, 1/4000) and anti-GAPDH (GeneTex, GT239, 1/10000) were used as a reference protein. Horse radish peroxidase (HRP)-conjugated goat anti-mouse (Sigma-Aldrich A4416, 1/5000) and HRP-conjugated goat anti-rabbit (Sigma-Aldrich A6154, 1/5000) were used as secondary antibodies. Proteins were detected by chemiluminescence with Immobilon western chemiluminescent HRP substrate (Millipore, WBKLS0100) using a western blot Imager (Bio-Rad, ChemiDocTM XRS +). Quantification was done with Fiji image processing freeware [29 (link)] by measuring the intensity of each band in a rectangular selection of fixed size and the intensity of each marker band was expressed relative to that of the corresponding reference protein in the same lane.