Lamin b antibody

Immunofluorescence staining of micronuclei was performed to confirm their presence after X-ray irradiation in HOC313-LM cells. HOC313-LM were fixed in 2% formalin, permeabilized with 0.2% Triton X-100, treated with blocking solution (1% bovine serum albumin in phosphate-buffered saline), and then incubated with Lamin B antibody (1:100, sc-6216, Santa Cruz Biotechnology) for 1 hour. Alexa Fluor 488-conjugated antibody was utilized as a second antibody (1:100, A-11094, Thermo Scientific). DAPI (1:1000, D1306, Thermo Scientific) and rhodamine phalloidin (1:1000, R415, Thermo Scientific) were used to stain nuclei and F-actin, respectively.

Nuclear proteins were isolated from iris and ciliary body by using ReadyPrepTM Protein Extraction Kit (Cytoplasmic/Nuclear, Bio-Rad, Hercules, CA, USA). Protein concentration was adjusted equally with protein assay kit (Bio-RAD, Hercules, CA, USA), then re-suspended in 5x sample loading buffer, heated for 5 min at 95°C and separated on 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a nitrocellulose membrane (AmershamTM HybondTM-ECL, GE Healthcare, UK), blocked with 5% Bovine albumin BSA (A9418, Sigma-Aldrich, USA), and incubated with NF-κBp65 antibody (1∶300, sc-372, Santa Cruz Biotechnology, INC.) and Lamin B antibody (1∶500, sc-6216, Santa Cruz Biotechnology, INC.) at 4°C overnight. After washing with TBS-0.05% tween-20 (TBST), HRP-coupled secondary antibodies (1∶1000, Santa Cruz Biotechnology, INC.) were applied to the membrane for 1 hour at room temperature, followed by three washes with TBST. The immunoreactive bands were visualized with enhanced chemiluminescence reagents (GE Healthcare, UK) and images were captured by the Universal Hood II image system (Bio-Rad Laboratories, Segrate, Italy). Band intensities of NF-κBp65 were normalized with those of internal control (Lamin B) using NIH Image J software (version 1.47).

Nuclear extracts from primary microglia were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) as previous described (61 (link)). Proteins were quantified by the BCA protein assay (Bio-Rad) and protein extracts were aliquoted and stored at −80°C. Rabbit anti-mouse NF-κB p65 antibody (Cell Signaling Technology) was used at a 1:1000 dilution. Lamin B antibody (1:400; Santa Cruz Biotechnology) and histone A3 antibody (1:2000; Cell Signaling Technology) were used for loading control normalization. After incubating with primary and secondary antibodies, the blots were detected with femto chemiluminescent substrates (Thermo Fisher Scientific).

Jurkat cells were electroporated with 80nM of siRNA against human RUNX1 using the Amaxa Nucleofector system (Lonza Inc., Allendale, NJ, USA). Multiple siRNA targeting RUNX1 mRNA (Dharmacon Inc, Lafayette, CO) were initially screened. Only the siRNA with greater than 50% knock down efficiency of RUNX1 protein, were used for subsequent experiment. Briefly, 1×10⁶ Jurkat cells were electroporated with either a control or two different RUNX1 siRNA (siR1, siR2) for 18 hours. Cells were harvested for either ChIP or western blot analysis.
Blots were probed with polyclonal RUNX1 antibody (Cell Signaling Technology Inc, Beverly, MA) or SCL antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). Blots were stripped and re-probed with polyclonal Actin, Lamin B antibody or mouse monoclonal GAPDH antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). Protein were detected using species matched HPR-conjugated secondary antibodies and chemiluminiscence imagining.

Pheophytin-b was purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. LPS (Escherichia coli O26:B6, L2654) was purchased from Sigma (St. Louis, MO, USA).
The NOS2 antibody was purchased from BD Biosciences (San Jose, CA, USA). Antibodies against p44/p42 MAP Kinase (Thr202/Tyr204), p38 MAP Kinase (Thr180/Tyr182), SAPK/JNK (Thr183/Tyr185), ERK1/2 MAP Kinase (Thr202/Tyr204), phospho-IκB-α (Ser32/36), STAT1 (Tyr701), Akt (Ser473), PI3K p85 (Tyr458)/p55 (Tyr199), c-Fos (9F6), c-Jun (Ser73), and COX-2 were all purchased from Cell Signaling (Danvers, MA, USA). The NF-κB p65 rabbit monoclonal antibody (Cell Signaling), lamin B antibody (Santa Cruz, CA, USA), and mouse monoclonal beta-Actin antibody (Abcam, Cambridge, MA, USA) were also utilized.