Mouse anti lamin a c

Protein lysates (12.5–50 µg for cytosolic fractions and 10–15 µg for nuclear fractions) in 1X SDS/β-mercaptoethanol buffer were resolved on a 4–20% TGS stain free gel (BioRad, Hercules, CA, USA) and electrotransferred onto a polyvinylidene difluoride transfer membrane. Western blot analysis was performed using mouse anti-lamin A/C (1:2000; Cell Signaling Technology #4777, Danvers, MA, USA), rabbit anti-SQSTM1/p62 (1:1000; Cell Signaling Technology #5114) or rabbit anti-GAPDH (1:1000; Cell Signaling Technology #5174) and horseradish peroxidase-conjugated goat anti-rabbit (1:2000; Cell Signaling Technology #7074) or goat anti-mouse (1:5000, Abcam #ab6789, Waltham, MA, USA) antibodies. Protein bands were visualized by chemiluminescence using a ChemiDocTouch Imaging System (BioRad, Hercules, CA, USA). Bands were quantified using the ImageLab software version 5.2.1.

Rabbit anti-human perilipin-3 antibody was raised against a peptide of human perilipin-3 segment (amino acids 305–318)^{47 (link)}. Rabbit anti-CCTα antibody was donated by Dr. Neale Ridgway (Dalhousie University). Rabbit anti-lamin B receptor (Genway Biotech GWB-C7CA28), rabbit anti-lamin B1 (Abcam ab16048), mouse anti-lamin A/C (Cell Signaling #4777), rabbit anti-actin (Sigma A2066), and mouse anti-FLAG (Sigma F1804), goat anti-MTP (Santa Cruz sc-33116), mouse anti-ApoE (Immunogenetics M-012-0500), mouse anti-choline kinase β (Santa Cruz sc-398957), mouse anti-diacylglycerol cholinetransferase 1 (Santa Cruz sc-515577), rabbit anti-choline/ethanolamine phosphotransferase 1 (Abgent #AP10372a), goat anti-ApoB (Rockland 600-101-111), rabbit anti-ApoC-III (Thermo Fisher 6H21L11), mouse anti-V5 (Thermo Fisher R960-25), mouse anti-choline kinase α (CKα) (Proteintech 13520-1-AP), guinea pig anti-perilipin-3 (Progen GP30), and mouse anti-nucleopore complex (Covance MMS-120P) antibodies were obtained from the respective suppliers. Secondary antibodies conjugated to fluorochromes and peroxidase were purchased from Thermo Fisher, Jackson ImmunoResearch Lab, and Bethyl Laboratories. See Supplementary Table 1 for dilutions of antibodies used for immunofluorescence labeling and Western blotting.

Tissue sections were stained with hematoxylin and eosin (H&E). Mast cells were stained with 1% toluidine blue^{21 (link)}. T cells were labeled by with rabbit anti-CD3 (1:800, Glostrup, Denmark). Other immunolabelings were done with rabbit anti-histone H3 antiserum (1:50, Cell Signaling Technology, Danvers, MA) and mouse anti-lamin A/C (1:200, Cell Signaling Technology, Danvers, MA). Secondary antibodies conjugated to Alexa Fluor 546 fluorescent dye (Molecular Probes, Leiden, Netherlands) were used at a dilution of 1:500. When the primary antibodies were replaced by unrelated control antibodies, the fluorescence signals were abolished, confirming the specificity of the immunolabelings.

Cells were rinsed in PBS at 37 °C, fixed in 4% paraformaldehyde for 15 min at 37 °C, permeabilized (PBS containing 0.1% Triton X-100) for 5 min, blocked (PBS containing 2% bovine serum albumin and 2% normal goat serum) for 30 min, and incubated with the following antibodies at the indicated dilution: mouse anti-α-tubulin (1:2,000; T6199; Sigma) and mouse anti-lamin A/C (1:200; 4777, Cell Signaling Technology). Secondary antibodies conjugated to Alexa Fluor 488 and 568 (Molecular Probes) were used at 1:2,000 dilution. After a 5-min wash in PBS containing 4′,6-diamidino-2-phenylindole (DAPI), the coverslips were mounted in ProLong Diamond (Thermo Fisher).