Anti lamin a c

The cells were lysed with lysis buffer containing 50 mM Tris–HCl, pH 7.4, 0.1% SDS, 0.25 mM deoxycholate, 150 mM NaCl, 2 mM EGTA, 0.1 mM Na₃VO₄, 10 mM NaF, 1 mM PMSF, and complete Protease Inhibitor (Roche). The lysates were then sonicated and centrifuged to collect the supernatant. Immunoprecipitation was carried out using an anti-PER2 antibody overnight at 4 °C. Protein A (Pierce) was used to collect the antibody-protein complex and precipitates were washed three times with lysis buffer. Lysates and immunoprecipitates were separated by SDS-PAGE and Immunoblotting was performed as described previously8 (link). We used anti-PER24 (link), anti-BMAL1 (abcam), anti-CLOCK (Thermo Scientific), anti-Lamin A/C (BD Bioscience), anti-GAPDH (Ambion), anti-HA (Cell signaling), anti-FLAG (Sigma), and anti-mPML (Upstate) antibodies.

RPMI, DMEM, and FBS were from Invitrogen. Hygromycin B and puromycin were from Gibco. Primary antibodies used were anti-HA (Cell Signaling Technology, catalog number C29F4), anti-pHistoneH3 (S10) (Abcam, catalog number ab14955), anti-LaminA/C (BD Biosciences, catalog number 612162), anti-GAPDH (Sigma-Aldrich, catalog number CB1001), and anti-β-actin (Santa Cruz Biotechnology, Inc., catalog number sc-1616). Secondary HRP-conjugated antibodies used were goat anti-rabbit (Cell Signaling Technology, catalog number 7074S) horse anti-mouse (Cell Signaling Technology, catalog number 7076S) and donkey anti-goat (Santa Cruz Biotechnology, Inc., catalog number sc-2020). Chemiluminescence reagent for Western blotting was SuperSignal™ West Pico PLUS. Mounting media for fluorescence microscopy was ProLong® Gold Antifade Reagent with DAPI. LPA supplied in chloroform was from Avanti Polar Lipids {Acyl-linked 18:1 lysophosphatidic acid [1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt)]}. For sulfenylation studies, DCP-Bio1 was from by Xoder Technologies (Winston Salem, NC), sepharose CL-4B resin was from Sigma, and high capacity streptavidin agarose resin was from Thermo Scientific. Propidium iodide and the nucleotides GTP and dATP were from Invitrogen.

Reagents and antibodies used in this study were purchased from miscellaneous sources. Cycloheximide (MP Biomed, # 100183) was used at 1 µg/ml for different time points. MG132 (HiMedia, 474787-10MG) was used at 10 µM concentration for 8 h. The antibodies used for western blotting are anti-Nup88 (BD Biosciences, # 611896, 1:2000 dilutions), anti-Nup62 (BD Biosciences, # 610497, 1:6000 dilutions), anti-GAPDH (Abgenex, # 10-10011, 1:6000 dilutions), anti-GFP (Santa Cruz, # 9996 1:5000 dilutions), anti-GST (1:500 dilutions), anti-HA (Sigma, # H6908, 1:2000 dilutions), anti-FLAG (Sigma, # F7425, 1:2000), anti-Actin (BD Biosciences, # 612656, 1:5000 dilutions), anti-Lamin A/C (BD Biosciences, # 612162, 1:2000 dilutions), anti-NFκB p65 (BD Biosciences, # 610868, 1:2000 dilutions), goat anti-rabbit IgG-HRP (GeNei, # 114038001A, 1:10000 dilutions), goat anti-mouse IgG-HRP (GeNei, # 114068001A, 1:10000 dilutions). A polyclonal antibody was generated against Nup88 by immunizing rabbits with Nup88 fragment (aa 1 - 584) lacking the C-terminal coiled coil-domain as an antigen. Antibodies were purified from immunized serum over NHS-Sepharose beads immobilized with appropriate antigen.