Anti emerin

ES cells were treated with 1 μM RA for 4 days. The cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein concentration was measured by BioRad-DCC protein assay. The lysates were diluted 1:1 with BioRad 2X loading buffer, and equal protein was loaded on 4-12% Tris glycine-polyacrylamide (Invitrogen) gels. Separated protein was electrotransferred to nitrocellulose membrane (Pall, Pensacola, FL), and immunoblotted with primary antibodies. Antibodies used were: mouse monoclonal anti-lamin A/C (1:1000, Active Motif, #39288); rabbit polyclonal anti-emerin (1:1000, ProteinTech, #10351-1-AP); mouse monoclonal anti-actin (1:5000, BD-Transduction, #612656); rabbit polyclonal anti-ERK2/1 (1:1000, Cell Signaling, #9102); mouse monoclonal anti-Dab2 (1:2000, BD-Transduction, #610464); rabbit polyclonal anti-Gata4 (1:1000, Santa Cruz, sc9053); mouse monoclonal anti-Oct3/4 (1:1000, Santa Cruz, sc5279); rabbit anti-Nanog (1:1000, Abcam, ab80892); mouse monoclonal anti-N-cadherin (1:1000, BD-Transduction, #610920). HRP-conjugated secondary antibodies were from BioRad and used at 1:5000 dilution. SuperSignal West Dura Extended Duration substrate (Thermo Scientific, #34076) was used to visualize the immunoblots.

An antibody against GAPDH (MAB374) was from Millipore; Calnexin (ab22595) was from Abcam; Tubulin (DM1A) was from Sigma; CHMP2A (10477–1-AP) was from Proteintech; IST1 (51002–1-AP) was from Proteintech; CHMP7 (16424–1-AP) was from Proteintech; GFP (7.1/13.1) was from Roche; mCherry (ab167453) was from Abcam; HA.11 (16B12) was from (Biolegend); γH2AX (05–636) was from Sigma; 53BP1 (NB100-305) was from (Novus Biologicals). Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S). Anti-Histone H3 pS10 was from Cell Signaling Technology (9701S). Anti-LEM2 was from Sigma (HPA017340); anti-HSP90 (F8) was from Santa Cruz; anti-Emerin (10351–1-AP) was from Proteintech. Anti-LAP1 (21459–1-AP) was from Proteintech. Alexa conjugated secondary antibodies were from Invitrogen and HRP-conjugated secondary antibodies were from Millipore. IRDye 800 CW (925–32210) and IRDye 680 RD (925–68071) were from LI-COR Biosciences. Anti-peptide antibodies against CHMP7 pSer3 (3891 and 3892) were generated by immunisation of rabbits with KLH-conjugated peptides (MWpSPEREAEAPAGGC) by GenScript Biotech (Netherlands) B.V.