Anti flag tag f3165

Antibodies employed were: anti-PCAF (sc-13124) from Santa Cruz (Dallas, TX, USA) used 1:50 for immunofluorescence (IF) or 1:100 for western blotting (WB); anti-lamin A/C, goat polyclonal (SC-6215) from Santa Cruz (Dallas, TX, USA) used at 1:100 dilution for and in situ proximity ligation assay (PLA); anti-HDAC2, rabbit polyclonal (AB16032) from Abcam (Cambridge, UK) used at 1:2000 for WB and 1:200 for PLA analysis; anti-H3K9 acetylated, rabbit polyclonal (07-352) from Merck Millipore (Milan, Italy) used at 1:200 for IF; anti-FLAG tag (F3165) from Sigma (St.Louis, MO, USA) and anti-HA tag (sc-7392) from Santa Cruz (Dallas, TX, USA) were used at 1:1000 in WB and 1:300 in IF; anti-GFP (sc-9996) from Santa Cruz (Dallas, TX, USA) was used at 1:500 in WB; anti acetyl-lysine (9824S) from Cell Signaling (Leiden, The Netherlands) was used at 1:2000 in WB; anti myogenin from Santa Cruz (Dallas, TX, USA) was used at 1:100 in IF.
A total of 2 μM of Mevinolin (Sigma, St. Louis, MO, USA), a drug able to induce non-farnesylated prelamin A accumulation through inhibition of farnesyl production, was added to HEK293 cells for 18 h. A total of 100 μM of Sirtinol, a SIRT1 inhibitor, able to induce PCAF acetylation [31 (link)], was added to human fibroblasts for 18 h; while 50 μM of sirtuin 1 activators (MC 2562 and MC 2528) PCAF deacetylating drugs were added for 18 h.

Cells were seeded in 6-well plates on coverslips for 24 h. Cells were fixed in 4% paraformaldehyde for 15 min, followed by cell permeabilization with 0.2% TritonTMX-100 for 10 min. The cells were washed with PBS three times/ 5 min. Cells were blocked by 3% BSA for 10 min and incubated overnight at 4 °C in primary antibodies (anti-Flag tag, F3165, 1:10000, sigma) (anti-HA tag, H6908, 1:5000, sigma) diluted in 3% BSA. Secondary antibodies labeled with Alexa fluorophore 488/592 were diluted 1:500 in 3% BSA and incubated for 1 h at room temperature. Nuclear was staining with DAPI. Images were captured with a Leica laser confocal microscope.