Anti kiaa1199

Antibodies to ERK, phospho-ERK, p38, phospho-p38, JNK, phospho-JNK, NFκBp65, phospho- Ser276/Ser536NFκBp65, CREB, phospho-Ser133CREB, c-Fos, ATF2, phospho-Thr71ATF2, MSK1 and phospho-Thr581/Ser 376/Ser360MSK1, JAK2 and phospho-Tyr221JAK2, STAT3 and phospho-Tyr705/Ser727STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies to β-actin and HYBID (Anti-KIAA1199) were obtained from Sigma-Aldrich Corp., St. Louis, MO, USA). The antibody to protein tyrosine phosphatase, nonreceptor type 9 (PTPMEG2/PTPN9) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The NF-κB activation inhibitor II (JSH-23), the MEK inhibitor (U0126), and the STAT3 inhibitor were from Calbiochem (San Diego, CA, USA). The p38 inhibitor (SB239063) and the JNK inhibitor (JNK inhibitor II) were from Santa Cruz Biotechnology and Merck KGaA (Darmstadt, Germany), respectively. Okadaic acid and sodium orthovanadate were obtained from Calbiochem.

Western blot analyses were performed on cell lysates prepared from MDA-MB-231 and Hs578T cell lines as described previously [12 (link)]. Briefly, triplicate cell cultures were first washed with phosphate buffered saline (PBS, Invitrogen) and then lysed by mixing 1:1 with 2× sodium dodecyl sulphate sample buffer (100 mM Tris–HCl, pH = 6.8, 200 mM DTT, 4% SDS, 20% glycerol and 0.002% bromoplenol blue). Cell lysates were separated by 10% SDS-PAGE. Proteins were transferred to PVDF membranes (Immobilon 0.45 μm, Millipore, USA) and immersed in a blocking solution containing 5% non-fat milk and 0.1% Tween-20 for 1 h. The membranes were washed and incubated with primary antibodies (rabbit polyclonal anti-alpha-tubulin (abcam) at 1:1000 dilution, rabbit polyclonal anti-KIAA1199 (Sigma-Aldrich) at 1:100 dilution, rabbit ployclonal anti-KIAA1199 antibody (Protein Tech Group, Chicago, IL) at 1:800 dilution or rabbit anti-Caspase-3 (8G10) monoclonal antibody (Cell Signaling) at 1:1000 dilution) for 2 h and then with secondary antibodies for 1 h at room temperature. After washing the resulting bands were visualized using the standard ECL procedure, quantified by densitometry and normalized to the corresponding α-tubulin bands.