P erk1 2 9101s

Harvested cells were incubated with Radioimmunoprecipitation assay buffer (RIPA buffer) (50 mmol/L Tris-Cl (pH 7.5), 150 mmol/L NaCl, 0.5% [wt/vol] sodium deoxycholate, 1% NP-40 [vol/vol], 0.1% SDS [wt/vol] and 1 mmol/L EDTA) containing a protease inhibitor cocktail (1 μg/mL aprotinin and leupeptin) to extract proteins from the cells. Proteins in the cell lysate (30 μg) were separated through SDS-PAGE and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were blocked with 5% skim milk for 3 h before adding primary antibodies against pCDK1 (sc101654), cyclin B1 (sc245), MDR1 (sc55510), and HOXC6 (sc376330), which were purchased from Santa Cruz; PARP (9542S), AIF (5318P), ERK1/2 (9102S), and p-ERK1/2 (9101S) were purchased from Cell Signaling. After being washed twice, the membranes were incubated with the corresponding secondary antibodies (HRP-conjugated-linked anti-mouse IgG or HRP-linked anti-rabbit IgG) for 1 h (dilution ratio 1:5000). Protein signals were analyzed with a luminescent image analyzer (LAS-1000; Fujifilm, Tokyo, Japan) using the Immobilon ™ Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA).

Mouse kidneys were homogenized in SDS Laemmli buffer and loaded onto 10% SDS-polyacrylamide agarose electrophoresis gels essentially as described previously [27 (link), 47 (link)]. Primary antibodies pERK1/2 (9101 S), pAKT (4060 S), pSTAT3 (9145 S), STAT3 (9139 S), cyclinD1 (2978 S), S6 (2317 S), pS6 (4858 S), PDGFRβ (3169), VEGFR2 (9698), and FGFR1 (9740) from Cell signaling (Danvers, MA, USA); YAP (SC-101199), GAPDH (SC-32233), ERK1/2 (SC-94), cMyc (SC-40) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and α-SMA (ab5694; Abcam, Cambridge, MA, USA); and secondary antibodies, anti-mouse (P0447) and anti-rabbit (P0448) from Dako (Santa Clara, CA, USA) and ECL reagent from Amersham (GE Health care, Buckinghamshire, UK) were used.