P fos

Whole-cell lysates for western blotting were prepared as described [4 (link)], and protein lysates were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated overnight at 4°C with primary antibodies at the following dilutions: JUN, p-JUN, FOS, p-FOS, JUNB 1:500 (60A8, D47G9, 9F6, D82C12, C37F9, respectively; all from Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com); Mn-SOD2 1:500 (06-984; Millipore, Billerica, MA, http://www.millipore.com); p21 and p53 1:200 (sc-756, sc-99, respectively; Santa Cruz); β-actin 1:5000 (8226, Abcam, Cambridge, MA, http://www.abcam.com); p16 1:500 (554079; BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com); PKD/PKCμ, Phospho-PKD/PKCμ (Ser916), Phospho-PKD/KCμ (Ser744/748) 1:1000 (2052, 2051, and 2054, respectively; Cell Signaling Technology), were used for SER 910 and 938/942 detection respectively; SRF 1:500 (NBP1-61263; Novus Biologicals, Littleton, CO, http://www.novusbio.com). Signals were detected using the appropriate peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark, http://www.dako.com). Western blots were scanned and quantified by densitometry using ImageJ software (NIH, Bethesda, MD, www.nih.gov).