Alexa fluor 800 rabbit anti mouse

RAW264.7 cells were transfected with miR-124 mimic or All Star negative control (NC) siRNA and cultured as described above for 24 h with RANKL. Then, cells were lysed and total cellular proteins were extracted using RIPA buffer (Cell Signaling Inc., Beverly, MA, USA). Western blot was performed as previously reported [4 (link)]. Membranes were incubated overnight at 4 °C with the following antibodies: NFATc1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:1000 dilution; p-ERK1/2 and ERK1/2 (Cell Signaling Inc., Beverly, MA, USA) at 1:1000; and β-actin (Sigma Aldrich, St. Louis, MO, USA) at 1:5000. The secondary antibodies Alexa Fluor 680 Goat anti-Rabbit (1:2000) and Alexa Fluor 800 Rabbit anti-Mouse (1:5000) (Molecular Probes, Life Technologies, Carlsbad, CA, USA) were incubated for 1 h at room temperature. Protein visualization and quantization were carried out with the LI-COR Odyssey scanner and software (LI-COR Biosciences, Lincoln, NE, USA). Blots were imaged using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) according to the manufacturer’s instructions. Experiments were repeated two times.

A549 cells (1 × 10⁷ cells) were cultured in 10-cm dishes (Falcon) and allowed to adhere for 24 h. After treatment with BMS- 345,541 1 μM for 1 h, followed by incubation with LPS 100 ngr/ml for 4 h, the cells were washed twice with cold PBS. Whole-cell lysates were obtained using RIPA buffer (Cell Signaling Inc. Beverly, MA, USA). The protein concentration of cell lysates was determined by the Bradford method. An amount of protein (30 μg) was separated on 8–16% Tris–Glycine Gel (BioRad) gels by electrophoresis and transferred to a nitrocellulose membrane. The membranes were subsequently incubated for 1 h at room temperature with 3% BSA in TBS buffer (0.1% v/v) to block non-specific binding and incubated with an appropriate primary antibody in 1% BSA in TBST (tween 0.01% v/v). Antibodies polyclonal antimouse recognizing HSP90, IKKα/β, COX2, HO1 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibody polyclonal antirabbit recognizing p-IKKα/β was purchased from Cell Signaling Technology Massachussetts, USA. Incubation with the secondary antibodies Alexa Fluor 680 goat anti-rabbit (1:2000) and Alexa Fluor 800 rabbit anti-mouse (1:5000) (Molecular Probes, Life Technologies, Carlsbad, CA, USA) was performed for 1 h at room temperature. Densitometry analysis was conducted using the Odyssey Infrared Imaging System (LiCOR Bioscience, NE, USA).