PLoS Med. 2006 October; 3(10): e420.
Figure 3 Dysfunctional KEAP1朜RF2 Interaction in NSCLC Tumors (A) Immunohistochemical analysis of NRF2 in NSCLC tissues. Part a shows a patient (PT-18) with mutation in KEAP1 showing strong nuclear and cytoplasmic staining. Part b shows a patient negative for mutation (PT-28) showing weak cytoplasmic staining. Part c shows a patient negative for mutation (PT-20) showing increased nuclear and cytoplasmic staining in tumor tissue. Part d shows weakly staining normal bronchus from the same patient (PT-20). (B) Total GSH and enzyme activities of NQO1 and total GST in NSCLC and matched normal tissues. Raw data for the heat maps are presented in Table S4. *, samples harboring KEAP1 mutation; ? nmol/mg protein; ? nmol DCPIP reduced/min/mg protein; ? nmol of product formed/min/mg protein.
Am J Pathol. 2008 July; 173(1): 30?1.
Figure 1 HGF inhibits renal infiltration of inflammatory CD3-positive T cells. A朎: Immunohistochemical staining revealed an increased infiltration of CD3-positive T cells in the obstructed kidney at 7 (B) and 14 (D) days, respectively, after UUO, compared with sham control (A). Delivery of HGF gene effectively inhibited the infiltration of T cells in the obstructed kidneys at 7 (C) and 14 (E) days after UUO, respectively, as indicated. F: Graphic presentations of quantitative data are given. Data were presented as mean ?SEM of five animals per group (n = 5). *P < 0.01 versus sham control. ?/SUP>P < 0.01 versus pcDNA3. Scale bar = 20 靘. Am J Pathol. 2008 July; 173(1): 30?1.
Am J Pathol. 2008 July; 173(1): 30?1.
Figure 2 HGF inhibits renal infiltration of inflammatory F4/80-positive macrophages. A朎: Immunohistochemical staining revealed an increased infiltration of F4/80-positive macrophages in the obstructed kidney at 7 (B) and 14 (D) days, respectively, after UUO, compared with sham control (A). Delivery of HGF gene effectively inhibited the infiltration of macrophages in the obstructed kidneys at 7 (C) and 14 (E) days after UUO, respectively, as indicated. F: Graphic presentation of quantitative data are given. Data were presented as mean ?SEM of five animals per group (n = 5). *P < 0.01 versus sham control. ?/SUP>P < 0.01 versus pcDNA3. Scale bar = 20 靘.
The Journal of Neuroscience, April 16, 2008, 28(16):4172-4182
Immunoprecipitation and immunoblotting. Immunoprecipitation was performed to study phosphorylation of 18 kDa subunit of complex I, Fe-S subunit of complex III, and cytochrome oxidase IV (COX IV) subunit of complex IV phosphorylation in different experimental conditions. The total synaptosomal fraction (500 礸) was dissolved with 0.3% SDS, sonicated two times for 5 s each, and then immediately adjusted to 0.1% of the SDS concentration in "immunobuffer" consisting of 50 mM Tris-HCl, 1% Triton X-100, 1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and 0.5% NP-40, pH 7.4, and incubated for 20 min at room temperature. The immunoprecipitation was performed using protein A-Sepharose beads (Sigma-Aldrich) and anti-phospho-threonine, anti-phospho-serine, and anti-phospho-tyrosine mouse monoclonal antibodies (Sigma-Aldrich) per the manufacturer's instructions. The samples were washed twice with immunobuffer after overnight incubation of samples with protein A-Sepharose beads (Sigma-Aldrich) and anti-phospho-threonine, anti-phospho-serine, or anti-phospho-tyrosine. The resulting pellet was used for immunoblotting using anti-18 kDa subunit of complex I, anti-Fe-S subunit of complex III, or anti-COX IV subunit of complex IV antibody. To determine the levels of PKC, PKC, PKC, cytochrome c, synaptophysin, and ?actin, 40 礸 of protein from either synaptosomal or soluble fraction was separated using 12% PAGE. The immunoblotting was performed using anti-PKC, anti-PKC, anti-PKC, anti-cytochrome c, anti-synaptophysin, and/or anti-?actin antibodies. Immunoreactivity was detected using enhanced chemiluminescence (ECL Western blotting detection kit; GE Healthcare, Little Chalfont, Buckinghamshire, UK). Western blot images were digitized at eight-bit precision by means of a CCD-based camera (8?2 bits) (Xillix Technologies, Vancouver, British Columbia, Canada) equipped with a 55 mm Micro-Nikkor lens (Nikon, Tokyo, Japan). The camera was interfaced to an image acquisition and analysis system (MCID model M2; Imaging Research, St. Catherines, Ontario, Canada). The digitized immunoblots were subjected to densitometric analysis using MCID software.
The Journal of Neuroscience, January 2, 2008, 28(1):50-59
Immunoprecipitation. Capture antibodies (5 礸) were cross-linked to protein A agarose using sulfo-SMCC (5 礸; Pierce, Rockford, IL), washed, and blocked with 1% BSA for 30 min at room temp, before incubating with cell lysates (750 礸) overnight at 4癈. Precipitated proteins were washed using spin columns (Sigma), eluted by boiling in gel loading buffer, then subject to PAGE as for immunoblotting.
