PLoS Med. 2006 October; 3(10): e420.

Figure 4 Status of KEAP1 and NRF2 Is Altered in Cancer Cells (A) Immunoblot showing increased nuclear localization of NRF2 in nuclear extracts (NE) from cancer cells. Cancer cells showed lower levels of KEAP1 (~69 kDa) and higher levels of NRF2 (~110 kDa) in total protein lysates (TP). NIVT and KIVT indicate NRF2 and KEAP1 in vitro transcribed/translated product, respectively. (B and C) Quantification of NRF2 and KEAP1 protein in immunoblots. For band densitometry, bands in nuclear extract blot (B) were normalized to Lamin B1, and those in total protein (C) were normalized to GAPDH. (D) Heat map showing relative expression of KEAP1, NRF2, and NRF2-dependent genes by real-time RT-PCR. Raw data for the heat maps are presented in Table S5.

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 4 HGF blocks IL-1β- or TNF-α-mediated RANTES expression in tubular epithelial cells. A: HGF blocked RANTES expression induced by IL-1β and TNF-α. Human proximal tubular epithelial cells (HKC-8) were treated with IL-1β (5 ng/ml), TNF-α (2 ng/ml), HGF (40 ng/ml), or in different combinations as indicated for 48 hours. Whole cell lysates were immunoblotted with antibodies against RANTES and actin, respectively. B: HGF inhibited IL-1β-induced RANTES expression in a dose-dependent manner. HKC-8 cells were incubated with IL-1β (5 ng/ml) alone, or with IL-1β plus increasing amounts of HGF as indicated. C: HGF inhibited IL-1β-mediated RANTES secretion. The supernatant of the HKC-8 cells after various treatments as indicated was immunoblotted with anti-RANTES antibody. D: HGF inhibited TNF-α-induced RANTES secretion. The levels of RANTES secreted in the supernatant of HKC-8 cells after different treatments were measured by a specific enzyme-linked immunosorbent assay.

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 5 Induction of RANTES expression is dependent on NF-κB signaling. HKC-8 cells were treated with IL-1β in the absence or presence of NF-κB signal inhibitor (20 μmol/L NF-κB SN50) for various periods of time as indicated. Whole cell lysates were immunoblotted with antibodies against RANTES and actin, respectively.

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 6 HGF does not affect IL-1β-mediated phosphorylation of IκBα and p65 NF-κB, as well as the degradation and abundance of IκBα and IκBβ. HKC-8 cells were treated with IL-1β (5 ng/ml) alone or IL-1β plus HGF (40 ng/ml). At different time points as indicated, cells were harvested and subjected to immunoblotting with antibodies against p-IκBα, IκBα, IκBβ (Α), p-p65 NF-κB, and p65 NF-κB (B), respectively. C: Long-term incubation with HGF also did not affect the phosphorylated status of IκBα and p65 NF-κB in HKC-8 cells.

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 8 Suppression of the RANTES expression by HGF is mediated by a PI-3-kinase-dependent pathway. A: Inhibition of PI-3-kinase by wortmannin blocked Akt phosphorylation, a downstream event of PI-3-kinase activation. HKC-8 cells were pretreated with wortmannin (10 nmol/L) for 30 minutes, followed by incubation with HGF for various periods of time as indicated. Whole cell lysates were immunoblotted with antibodies against p-Akt and total Akt, respectively. B: Inhibition of PI-3-kinase abolished HGF action and restored IL-1β-mediated RANTES expression. HKC-8 cells after various treatments as indicated were subjected to immunoblotting with antibodies against RANTES and actin, respectively.

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 9 HGF induces GSK-3β phosphorylation and its subsequent inactivation through PI-3-kinase-dependent pathway, and inhibition of GSK-3β suppresses RANTES induction. A: HGF induced GSK-3β phosphorylation in a PI 3-kinase-dependent manner. HKC-8 cells were pretreated with wortmannin (10 nmol/L) for 30 minutes, followed by incubation with HGF (40 ng/ml) for various periods of time as indicated. Cell lysates were immunoblotted with antibodies against p-GSK-3β and total GSK-3β. B: Inhibition of GSK-3β induces its phosphorylation. HKC-8 cells were treated with GSK-3β-specific inhibitors (5 μmol/L SB216763 and 30 mmol/L LiCl). Cell lysates were immunoblotted with antibodies against p-GSK-3β and GSK-3β, respectively. C: Inhibition of GSK-3β abolishes IL-1β-mediated RANTES expression. HKC-8 cells were treated with IL-1β for 48 hours in the absence or presence of GSK-3β inhibitors (30 μmol/L SB415286, 5 μmol/L SB216763, and 30 mmol/L LiCl). Cell lysates were immunoblotted with antibodies against RANTES and actin, respectively. D: Ectopic expression of GSK-3β enhanced TNF-α-mediated RANTES expression and primarily abolished the HGF-mediated inhibition of RANTES. HKC-8 cells were transfected with GSK-3β expression plasmid or control pcDNA3, and then treated with TNF-α or/and HGF as indicated. Am J Pathol. 2008 July; 173(1): 30–41.

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 10 HGF does not block IL-1β-mediated TNF-α expression which is independent on NF-κB signaling. A: HGF blocked IL-6 expression induced by IL-1β or TNF-α, but has no effect on TNF-α induction. HKC-8 cells were treated for 48 hours with IL-1β (5 ng/ml), TNF-α (2 ng/ml), HGF (40 ng/ml), or in combination as indicated. Whole cell lysates were immunoblotted with antibodies against IL-6 and TNF-α, respectively. Equal loading of the samples in each lane was confirmed by reprobing with actin (see Figure 3A). B: Induction of TNF-α expression by IL-1β in tubular epithelial cells was independent on NF-κB signaling. HKC-8 cells were pretreated with NF-κB SN50 for 30 minutes, followed by incubation with IL-1β for 24 and 48 hours, respectively. C: Blockade of GSK-3β did not affect IL-1β-mediated TNF-α expression. HKC-8 cells were treated with various GSK-3β inhibitors (30 μmol/L SB415286, 5 μmol/L SB216763, and 30 mmol/L LiCl) for 30 minutes before addition of IL-1β. Cell lysates were immunoblotted for TNF-α and actin, respectively. Am J Pathol. 2008 July; 173(1): 30–41

Reproductive Biology and Endocrinology 2008, 6:49

Effects of FSH and GDF-9 on PBX2 in SVOG cells. The cells were treated with FSH (100 ng/ml) or/and GDF-9 (50 or 100 mg/ml). For co-treatment, cells were pre-treated with FSH for 4 hr and then treated with GDF-9 for 24 hr. Ota et al. Reproductive Biology and Endocrinology 2008 6:49 doi:10.1186/1477-7827-6-49

Reproductive Biology and Endocrinology 2008, 6:44

Western blot analysis Western blot analysis was performed as described previously [20]. Briefly, cells were grown in 60 mm dishes and treated with vehicle (control), PDBu and IFNT (50 ng and 1000 ng), alone and in combination for 6 h as described above. Cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% SDS, 0.5% sodium deoxycholate, 1 mM DTT, 100 μM PMSF, 1% NP-40), and 1 × protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Lysates from triplicate treatment wells were combined, sonicated and centrifuged (10,000 g, 20 minutes) and supernatants were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Non-specific binding sites were blocked with 5% non-fat dry milk and membranes were incubated with antibodies to PLA2G4A, PLA2G4C, and PLA2G6 in 1:800, 1:400 and 1:1000 dilutions, respectively, overnight at 4°C. Membranes were rinsed 3 times and protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Arlington Heights, IL). A blot was prepared from each of the replicated experiments.

J. Clin. Invest. 118(1): 79-88 (2007)

Immunoblotting analyses. MACS-sorted B cells and MEFs were lysed in RIPA buffer and proteins quantified using a BCA assay (Pierce Biotechnology). Proteins (25 or 50 mg/lane) were electrophoretically separated on 4%–12% SDS-PAGE gels (Invitrogen), transferred to membranes (Protran; Schleicher and Schuell), and blotted with antibodies specific for β-actin, Puma (Sigma-Aldrich), c-Myc, p62 (Santa Cruz Biotechnology Inc.), p53 (BD Biosciences — Pharmingen; IC12, Cell Signaling), p19Arf (kindly provided by Charles Sherr, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA), cathepsin D (R&D Systems), and LC3 (polyclonal antibody raised in rabbits). Following incubation with primary antibodies, the blots were then incubated with appropriate anti-mouse, anti-rat, or anti-rabbit immunoglobulin secondary antibodies (Amersham). Bound immunocomplexes were detected by enhanced chemiluminescence (Amersham) or ECL SuperSignal (Pierce Biotechnology).