Pakt 9275

In order to investigate the effects of Chalcone T4 on the phosphorylation of intracellular signaling pathways NF-ĸB, p38 and ERK (MAPK), and AkT, macrophages were plated at a concentration of 3 × 10⁵ cells/well with α-MEM supplemented, and after 24 h de-induced with culture medium containing 0.3% FBS for 6 h, and then stimulated with RANKL for 10 and 40 min, with and without pretreatment with Chalcone T4. At the end of the experimental period, the medium was aspirated, the plates washed with PBS and RIPA Buffer (Sigma-Aldrich, San Luis, MO, USA) supplemented with phosphatase and protease inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA, USA) added to each plate. Total proteins were quantified by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). To perform the Western Blot, 20 μg of total protein per sample were used. The membranes were incubated with the primary antibodies (p-p38 #9211 Cell Signaling, p38 #9212 Cell Signaling, p-Akt #9275 Cell Signaling, Akt #9272 Cell Signaling, p-NF-kB #3033 Cell Signaling, NF-kB #sc8008 Santa Cruz, p-ERK #4376 Cell Signaling and ERK #9102 Cell Signaling) overnight. The results were obtained by densitometric analysis of the bands and normalized through the ratio between the phosphorylated and non-phosphorylated form of the target protein.

Western blots were performed as described in previous studies [3 (link),20 (link)]. Basically, 30–40 μg of the total lysate proteins were resolved through 8–12% SDS-PAGE and transferred to a PVDF membrane (GE Healthcare, Amersham, UK). Nonspecific protein binding was blocked with 5% skimmed milk in Tris-buffered saline-Tween- 20 (TBS-T) for 1h at RT. The membranes were probed with respective primary antibodies Santa Cruz, CA, USA—Akt1 (sc-5298), Bcl-2 (sc-7382), COL1A1 (sc-28657), Fas L (sc-956), Bad (sc-8044), GAPDH (sc-25778), tPA (sc-5239), and uPA (sc-14019); Cell Signaling Technology, Inc, pAkt (#9275), and c-Cas 3 (#9664). All the secondary antibodies (anti-rabbit, mouse and goat, HRP-conjugated) were purchased from Invitrogen (Carlsbad, CA, USA). Subsequent to the usual washing steps, the membranes were thereafter incubated with HRP conjugated secondary antibodies (1:10,000, GE Healthcare, Amersham, UK) and developed with ECL Substrate (Millipore, Billerica, MA, USA) and finally analysed with the LAS-3000 fluorescence imaging system (Fuji Film, Tokyo, Japan).The densitometry analysis of protein expression was performed though Image J software (NIH, Bethesda, MA, USA).