Mouse monoclonal anti β actin antibody

Nigericin and anti-β-actin (mouse) monoclonal antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS was prepared by A. actinomycetemcomitans as described previously [25 ,26 (link)]. LY294002 was purchased from MedChem Express (Monmouth Junction, NJ, USA). The JNK Inhibitor II and PD98059 were purchased from Merck Millipore (Billerica, MA, USA). SB239063 was purchased from Calbiochem (San Diego, CA, USA). Mg(OH)₂ nanoparticles were prepared in the same way as previously described [20 (link)]. Anti-IL-1β (rabbit) monoclonal, anti-IκBα (rabbit) polyclonal, anti-NF-κB p65 (rabbit) monoclonal, anhosphorpho-p38 MAPK (rabbit) monoclonal, anti-p38 MAPK (rabbit) polyclonal, anti-phospho-ERK1/2 (rabbit) monoclonal, anti-ERK1/2 (rabbit) monoclonal, anti-phospho-JNK (rabbit) polyclonal, anti-JNK (rabbit) polyclonal, anti-phospho-Akt (rabbit) monoclonal, and anti-Akt (rabbit) polyclonal antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). An anti-β-actin (mouse) monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-lamin B1 (rabbit) polyclonal antibody was purchased from Proteintech Biotechnologies (Rosement, IL, USA).

Spleens were harvested from mice and CD4⁺ T cells or total B cells were purified from single-cell suspensions by magnetic cell sorting using the appropriate MACS Cell Isolation Kit according to the manufacturers protocols (Miltenyi Biotec). The cell purity was routinely >90% pure following specific cell isolation (as determined by FACS analysis). Cells were lysed by incubation in 1X SDS-PAGE sample buffer for 3 min at 95°C followed by brief sonication to shear DNA. SDS-PAGE and immunoblotting was performed as described [23 (link)]. The amount of SNAP-23 protein present in cell lysates was determined by immunoblotting using a rabbit anti-mouse SNAP-23 antibody [3 (link)] and quantitated on a Molecular Dynamics Densitometer and ImageQuant software (Sunnyvale, CA). The loading of the different samples was normalized by re-probing every blot with mouse monoclonal anti-β actin antibody (Millipore).

Cell lysates were quantified for protein content using bicinchoninic acid (BCA) protein assay kit (Pierce Chemical, Rockford, IL). Equivalent amount of proteins were used for Western blot analysis as described previously (11). Protein bands were visualized by chemiluminescence using Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA). The following antibodies were used for detection of HCMV proteins: mouse anti-IE1 and IE2, (MAb810); mouse anti-UL44 (Santa Cruz biotechnology); mouse monoclonal anti-pp65 (Vector laboratories, Burlingame, CA); mouse monoclonal anti-β-actin antibody (Millipore, Billerica, MA); rabbit polyclonal anti-RPS14 and mouse monoclonal anti-MDM2 (AbCam, Cambridge, UK), mouse monoclonal anti-p53 (Santa Cruz); horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling); and HRP-conjugated sheep anti-mouse IgG, (GE Healthcare, Waukesha, WI). For detection of co-immunoprecipitated proteins, protein A-HRP conjugate (Cell Signaling Technologies, Beverly, MA) was used as secondary antibody to eliminate the interfering IgG bands [60 (link)].