Anti lamin b1 antibody

Whole cell and nuclear lysates were prepared by solubilizing whole cell and nuclear pellets in RIPA buffer, respectively. For co-immuno-precipitation experiments, proteins were extracted from the nuclear pellets using high-salt buffer (19 (link)). Target proteins were either immunoprecipitated or directly detected from total lysates after SDS-PAGE using specific antibodies according to manufacturers’ instructions. Antibodies specific for Lck, CRIF1 and epidermal growth factor receptor substrate 15 (Eps15) were purchased from Santa Cruz Biotechnology. Antibodies specific for phospho-Src family (Tyr416) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-lamin B1 antibody was purchased from Abcam (Cambridge, MA, USA). Appropriate secondary antibodies conjugated with horseradish peroxidase were used to detect signals by enhanced chemiluminescence system. For signal quantitation, the bands were digitalized and analyzed by ImageJ software.

48 h after siRNA transfection, the cells were washed with PBS buffer and lysed with RIPA buffer (1× PBS, 1% IP-40, 0.5% sodium deoxycholate, and 0.1% SDS). After 1 h incubation on ice, the cellular mixture was centrifuged, and the supernatant was collected. Equivalent amounts (∼30 μg) of prepared cellular extracts were separated on a 10% SDS–polyacrylamide gel and transferred to a PVDF membrane (Bio-Rad). The membranes were probed with antibodies against human Polλ (Bethyl Lab) or Flag (Sigma-Aldrich), followed by appropriate secondary antibodies conjugated with horseradish peroxidase. The signals were detected using ECL-Plus (GenDEPOT). For the loading control, anti-β-tubulin antibody (Santa Cruz Biotechnology) or anti-lamin B1 antibody (Abcam) was used.

Nuclear acid proteins were separated on 18 % polyacrylamide SDS-PAGE gel while nuclear extract were separated on 10 % polyacrylamide SDS-PAGE gel and transferred to a nitrocellulose membrane according to the protocol described by Towbin et al., 1979 [12 (link)]. The membrane was exposed to Ponceau S to verify the efficiency of the transfer. The membrane was blocked with 5 % milk in PBS buffer- Tween-20 0.05 % (PBS-T) for 2 h at room temperature and then incubated with the anti-histone H3 antibody (Abcam ab1791) 1:5000, anti-histone H4 antibody (Santa Cruz sc-8658-R) 1:2000, anti-acetyl-histone H4 (Millipore 06-866) 1:4000, anti-histone H4 acetyl K12 antibody (Abcam ab61238) 1:2000, anti-histone H4 mono methyl R3 antibody (Abcam ab17339) 1:500, anti-trimethyl-histone H4 (Lys20) antibody (Millipore 07-463) 1:500, anti-lamin B1 antibody (Abcam ab16048) 1:5,000, anti-fibrillarin 1:3,000, anti-PRP6 1:500, all diluted with 2 % milk PBS-T overnight at 4 °C. The membranes were rinsed 3 times with PBS-T and incubated for 2 h with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific G-21234) 1:7,500 or goat anti-mouse IgG antibody (Thermo Fisher Scientific G-21040) 1:7,500 diluted in 2 % milk PBS-T. The antibody staining reaction on the membranes were developed by SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific 34095).

Anti-β-actin antibody, BSS (CAS 83-46-5), protease inhibitor cocktail, and phosphatase inhibitor cocktail 3 were purchased from Sigma (St Louis, MO). Anti-AMPKα1/2 antibody, anti-p-AMPKα1/2 antibody, anti-PGC-1 (H-300) antibody, anti-UCP3 (E-18) antibody, 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine (CC), and 6-ketocholestanol (kCh) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-COX antibody was purchased from Cell Signaling (Danvers, MA). Anti-lamin B1 antibody was purchased from Abcam (Cambridge, MA). Bio-Rad assay reagent was purchased from Bio-Rad Laboratories (Richmond, CA, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA).

Refined storax oil was purchased from Tianjin ZHONGXIN Pharmaceutical Group Limited by Share Ltd Darentang Pharmaceutical Factory and conformed to the standard of China Pharmacopeia (2010 version). Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), glucose-free DMEM, fetal bovine serum (FBS), HBSS, and 0.25% Trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). CytoTox-ONETM Homogeneous Membrane Integrity Assay and Cell Counting Kit-8 (CCK-8) were purchased from Promega (Madison, WI, USA) and Dojindo (Kumamoto, Japan), respectively. DCFH-DA and Trizol were obtained from Invitrogen (Eugene, USA). Penicillin and streptomycin were purchased from Beyotime (Shanghai, China). Anti-NF-κB p65, anti-p-IκBα antibody, anti-p-IKK antibody, and anti-β-actin antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-LaminB1 antibody, anti-iNOS antibody, anti-IL-1β antibody, anti-ICAM-1 antibody, and anti-VCAM-1 antibody were purchased from Abcam Technology (Cambridge, MA, USA). SYBR® Select Master Mix and High Capacity cDNA Reverse Transcription Kits were obtained from Applied Biosystems (Foster City, USA).

For detecting the NE, each 2-μl extract sample was gently mixed on a glass slide with 4 μl of the fixing solution (3.7% formaldehyde, 2 μg/ml Hoechst 33258, 50% glycerol, 80-mM KCl, 15-mM NaCl, 15-mM Pipes-KOH, pH 7.2) containing 10 μM of 3,3′-dihexyloxacarbocyanine iodide (Sigma-Aldrich) and squashed under a 22-mm × 22-mm square coverslip.
For detecting replication activity and immunofluorescence, each 10 μl extract sample was diluted with 90-μl of extraction buffer (EB) (100-mM KCl, 2.5-mM MgCl₂, 50-mM Hepes-KOH, pH 7.5), 11 μl of 37% formaldehyde was then added, incubated at RT for 10 min, 1 ml of EB was added further, which was loaded into a swinging bucket for collecting suspension culture cells (CS-2, Tomy). Nuclei were collected by centrifuge (500g, 5 min) onto poly-L-lysine–coated coverslip (IWAKI) through 0.5-ml of EB plus 30% sucrose layer. DNA replication was labeled with 1 μM of CF594-dUTP (Biotium). Nuclear lamin B1 was detected by sequential incubation with anti-lamin B1 antibody (ab16048, Abcam) and Alexa 594 anti-rabbit IgG (Thermo Fisher). The coverslips were washed with TBS-T, PBS, and dH₂O and mounted on glass slides with 3 μl of SlowFade Gold (Thermo Fisher) containing 2 μg/ml Hoechst 33258.