The effects of the polybasic region (PBR) and ERK-induced S/T phosphorylation on the nuclear localization of RhoA and Rac1.

<p><b>(A)</b> COS-7 cells were transiently transfected with expression constructs encoding GFP-RhoA, GFP-Rac1, GFP-Rac1-T108E_RhoA-PBR, GFP-RhoA-S88E/T100E_Rac1-PBR, GFP-RhoA_Rac1-PBR and GFP-Rac1_RhoA-PBR. Cells were either not treated or treated with EGF for 15 min. The localization of these wold type and mutant RhoA and Rac1 proteins was examined by fluorescence microscopy. Size bar = 20 μm. <b>(B)</b> Quantification of the data in (A). Each value is the mean of at least three experiments with at least 20 transfected cells counted for each experiment. The error bar is the standard error. *, p<i><</i>0.05 and **, p<0.01.</p

Phosphorylation of RhoA on ⁸⁸S and ¹⁰⁰T by active ERK1 <i>in vitro</i>.

<p><b>(A)</b> Phosphorylation of His-RhoA, His-Cdc42, and His-Rac1 by ERK1 <i>in vitro</i>. The phosphorylation of purified His-tagged Rho proteins by purified active ERK1 was performed with an <i>in vitro</i> ERK kinase assay kit in the presence of [γ-³²P]ATP as described in the Materials and Methods. ³²P was detected by autoradiography. <b>(B)</b> Quantification of the data from three independent experiments as described in (A). The intensity of the bands of ³²P was normalized against the intensity of the His-tagged protein loading. The error bar is standard error. * indicates p<0.05. <b>(C)</b> Phosphorylation of GST-RhoA and mutant proteins by purified ERK1 <i>in vitro</i>. The phosphorylation of GST-RhoA (5 μg) and various mutant GST-RhoA (5 μg) by purified active ERK1 was performed as described in (A). GST was used as a negative control. GST fusion protein loading was verified by Amido Black stain of the PVDF membrane. <b>(D)</b> Quantification of the data from three independent experiments as described in panel C. The intensity of the bands of ³²P was normalized against the intensity of the GST fusion protein loading. The error bar is standard error. * indicates p<0.05 and ** indicates p<0.01.</p

The effects of RhoA phosphorylation (⁸⁸S and ¹⁰⁰T) on RhoA interaction with ERK and on EGF-induced ERK phosphorylation.

<p><b>(A)</b> The effects of RhoA phosphorylation on its interaction with ERK. COS-7 cells were serum starved and treated with EGF (50 ng/ml) for 15 min. The cell lysates were incubated with GST-fused wild type and mutant RhoA proteins bound to glutathione-sepharose beads. ERK pulldowns were analyzed by immunoblotting with antibodies to ERK. GST fusion protein loading was verified by Amido Black staining of the nitrocellulose membrane. <b>(B)</b> The effects of RhoA phosphorylation on EGF-induced ERK phosphorylation. COS-7 cells were transfected with expression constructs encoding GFP-tagged wild type and mutant RhoA proteins and these proteins were overexpressed. After serum starvation, cells were stimulated with EGF for 15 min. ERK phosphorylation and activation was determined by immunoblotting cell lysates with antibodies to p-ERK and p-ELK1, respectively. The expression of GFP-RhoA wild type and mutant proteins was determined by immunoblotting with antibodies to GFP.</p

Interaction between RhoA and ERK is mediated by the RhoA D-site.

<p><b>(A)</b> Co-immunoprecipitation of ERK and p-ERK with RhoA. COS-7 cells expressing GFP-RhoA were stimulated with EGF as indicated. GFP-RhoA was IPed from cell lysates with antibodies to GFP, and the co-IPed ERK and phosphor ERK (p-ERK) were analyzed by immunoblotting with antibodies to ERK and p-ERK. <b>(B)</b> Interaction between ERK and GST-RhoA. Lysates of COS-7 cells, with or without EGF stimulation, were incubated with GST-RhoA or GST bound to glutathione sepharose beads. The sepharose beads were collected, washed and analyzed by immunoblotting with antibodies against p-ERK and ERK. GST/GST-RhoA fusion protein loading was verified by Amido Black staining of the nitrocellulose membrane. <b>(C)</b> The expression levels of endogenous RhoA in COS-7 cells and various breast cancer cell lines. Cells were lysed and the expression levels of RhoA were determined by immunoblotting with anti-RhoA antibody. α-tubulin was used as a loading control. <b>(D)</b> Interaction between endogenous RhoA and ERK in COS-7 and SKBR3 cells. Endogenous RhoA was IPed from lysates of COS-7 and SKBR3 cells by anti-RhoA antibody, and the co-IP of endogenous ERK was determined by immunoblotting with antibodies to ERK. α-tubulin was used as a loading control. <b>(E)</b> The effect of the RhoA D-site on the interaction between RhoA and ERK. Lysates of COS-7 cells (with or without EGF stimulation) were incubated with GST-RhoA or mutant GST-RhoA with its D-site deleted (GST-RhoAΔD) bound to glutathione agarose beads. The sepharose beads were then collected, washed and analyzed by immunoblotting with antibodies against p-ERK and ERK. GST/GST fusion protein loading was verified by Amido Black stain of the nitrocellulose membrane.</p

The effects of RhoA phosphorylation on the activation of RhoA.

<p><b>(A)</b> The activity of wild type and various mutant RhoA proteins in response to EGF. COS-7 cells were transfected with expression constructs encoding wild type and mutant GFP-tagged RhoA proteins. After serum starvation, cells were stimulated with EGF for 15 min. Cell lysates were incubated with a GST fusion Rhotekin Rho-binding domain (GST-RBD). The active RhoA proteins that bound to GST-RBD were determined by immunoblotting with antibodies to GFP. <b>(B)</b> Quantification of the data from (A). The GTP-GFP-RhoA protein intensity was normalized to the intensity of the expressed GFP proteins (input) as detected by anti-GFP antibodies. <b>(C)</b> The effects of U0126 on the activation of endogenous RhoA in response to EGF. COS-7 cells were stimulated with EGF for the indicated time with or without U0126. The amount of active RhoA was determined by GST-RBD pull down assay as described in (A), except that antibodies to RhoA were used to detect the endogenous RhoA. <b>(D)</b> Quantification of the data from (C). The GTP-RhoA protein intensity was normalized to the intensity of the total endogenous RhoA protein (input) as detected by anti-RhoA antibodies. Each value is the average of at least three experiments and the error bar is standard error. * indicates p<0.05.</p

The effects of RhoA phosphorylation on its interaction with ROCK1 and mDia.

<p><b>(A)</b> Co-IP of wild type and mutant RhoA with ROCK1/mDia. COS-7 cells were transfected with constructs encoding wild type or mutant GFP-tagged RhoA and stimulated with EGF (50 ng/ml). GFP-tagged wild type and mutant RhoA proteins were immunoprecipitated from cell lysates with anti-GFP antibodies and the co-IPed ROCK1 and mDia were detected with anti-ROCK1 and anti-mDia antibodies. The input GFP was determined by immunoblotting the whole lysate with anti-GFP antibodies (bottom panel). (<b>B&C</b>) Quantification of the co-IPed ROCK1 (B) and mDia (C). The binding between ROCK1/mDia and the RhoA proteins was measured as the ratio of the ROCK1/mDia band intensity relative to the RhoA band intensity. Each value is the mean of at least three experiments. The error bar is standard error. * indicates p<0.05. (<b>D</b>) The effects of RhoA phosphorylation on ROCK1 activity. COS-7 cells were transfected with wild type or mutant GFP tagged RhoA. After pretreatment with Y-27632 (5uM) for 60 min, cells were stimulated with EGF (50 ng/ml) for 15min. (<b>E&F</b>) Quantification of the phosphorylation level of MYPT1 (E) and ROCK1protein (F) measured by the ratio between p-MYPT1 and ROCK1band intensity relative to GFP band intensity. Each value is the mean of at least three experiments. The error bar is standard error. * indicates p<0.05.</p

The effects of RhoA phosphorylation on its subcellular localization.

<p><b>(A)</b> Subcellular localization of GFP-tagged wild type and mutant RhoA by fluorescence microscopy. COS-7 cells were transfected with expression constructs encoding GFP-tagged wild type or mutant RhoA proteins. Cells were either untreated or treated with EGF for 15 min. The localization of various RhoA proteins was observed by fluorescence microscopy. Size bar = 20 μm. <b>(B)</b> Subcellular localization of wild type and mutant RhoA by subcellular fractionation. COS-7 cells were transfected with expression constructs encoding GFP-tagged wild type or mutant RhoA proteins. The transfected COS-7 cells expressing GFP-proteins were homogenized, and the cell homogenates were separated into nuclear and non-nuclear fractions as described in Materials and Methods. The loading volumes of the nuclear fraction and non-nuclear fraction were about 25% and 3% of total sample volume, respectively, and were analyzed by SDS-PAGE and immunoblotting. Nu, nuclear fraction; Non, non-nuclear fraction. <b>(C)</b> Quantification of the data in (B). <b>(D)</b> Subcellular distribution of endogenous RhoA. After EGF stimulation for 15 and 60 min, lysates of COS-7 cells were separated into nuclear, total membrane, and cytosolic fractions as described in Materials and Methods. One-third of the nuclear fraction, one-half of the membrane fraction, and 3% of the cytosolic fraction were analyzed by immunoblotting. <b>(E)</b> Quantification of the data in (D). Each value is the mean of at least three experiments. The error bar is standard error. * indicates p<0.05.</p

The effects of EGF and RhoA phosphorylation on actin stress fiber formation in COS-7 cells.

<p><b>(A)</b> Images of actin stress fibers. COS-7 cells were transfected with expression constructs encoding GFP-tagged wild type, 88A/100A (S88A/T100A) or 88E/100E (S88E/T100E) RhoA. The formation of actin stress fibers was viewed by fluorescence microscopy following staining with 70 nM rhodamine-conjugated phalloidin as described in the Materials and Methods. Boxed areas are shown at higher magnification. Size bar = 20 μm. <b>(B)</b> Quantification of the stress fibers was as described in the Materials and Methods. Each value is the mean of at least three experiments with more than 20 cells analyzed for each experiment. The error bar is standard error. * indicates p<0.05.</p

Synthesis of π-Extended Dithienobenzodithiophene-Containing Medium Bandgap Copolymers and Their Photovoltaic Application

<div><p>Two medium bandgap alternating conjugated copolymers, namely, poly{5,10-di(2-hexyldecyloxy)dithieno[2,3-<i>d</i>:2′,3′-<i>d</i>′]benzo[1,2-<i>b</i>:4,5-<i>b</i>′]dithiophene-2,7-diyl-<i>alt</i>-thiophene-2,5-di-yl} (<b>P1</b>) and poly{5,10-di(2-hexyldecyloxy)dithieno[2,3-<i>d</i>:2′,3′-<i>d</i>′]benzo[1,2-<i>b</i>:4,5-<i>b</i>′]dithiophene-2,7-di-yl-<i>alt</i>-thieno[3,2-<i>b</i>]thiophene-2,5-diyl} (<b>P2</b>), were prepared by the palladium-catalyzed Stille polycondensation and characterized by gel permeation chromatography (GPC), UV-Vis absorption and photoluminescence (PL) spectra, thermal gravimetric analysis (TGA), cyclic voltammetry (CV) <i>etc</i>. The resultant copolymers show moderate solubility in common organic solvents and enough stabilities for photovoltaic application. And both copolymers absorb the solar light from 300–600 nm, with the optical band gaps () calculated from the onset of absorption in the solid film of ca. 2.1 eV. The highest occupied molecular orbital (HOMO) levels of two copolymers determined by CV were at about −5.35 eV. Photovoltaic propertites of the polymers were investigated by using the polymers as donor and [6,6]-phenyl-C₇₁ butyric acid methyl ester (PC₇₁BM) as acceptor with a weight ratio of polymer:PC₇₁BM of 1:2. The power conversion efficiencies (PCEs) of polymer solar cells based on PDTBTT-TT reached 2.50%, with an open-circuit voltage (<i>V</i>_oc) of 0.70 V, a short-circuit current density (<i>J</i>_sc) of 6.89 mA cm⁻², and a fill factor (<i>FF</i>) of 52%, under the illumination of AM1.5, 100 mW cm⁻². These results indicate that dithieno[2,3-<i>d</i>:2′,3′-<i>d</i>′]benzo[1,2-<i>b</i>:4,5-<i>b</i>′]dithiophene (DTBDT) is a promising building block for the high-performance organic electronic materials.</p></div

Boosting Up Performance of Inverted Photovoltaic Cells from Bis(alkylthien-2-yl)dithieno[2,3‑<i>d</i>:2′,3′‑<i>d</i>′]benzo[1,2‑<i>b</i>:4′,5′‑<i>b</i>′]di thiophene-Based Copolymers by Advantageous Vertical Phase Separation

The photovoltaic cells (PVCs) from conjugated copolymers of PDTBDT-BT and PDTBDT-FBT with 5,10-bis­(4,5-didecylthien-2-yl)­dithieno­[2,3-<i>d</i>:2′,3′-<i>d</i>′]­benzo­[1,2-<i>b</i>:4,5-<i>b</i>′]­dithiophene as electron donor moieties and benzo­thiadiazole and/or 5,6-difluorobenzo­thiadiazole as electron acceptor moieties are optimized by employing alcohol-soluble PFN (poly­(9,9-bis­(3′-(<i>N</i>,<i>N</i>-dimethyl­amino)­propyl)-2,7-fluorene)-<i>alt</i>-2,7-(9,9-dioctyl­fluorene)) as cathode modification interlayer. The power conversion efficiencies (PCEs) of inverted PVCs (<i>i-</i>PVCs) from PDTBDT-BT and PDTBDT-FBT with devices configuration as ITO/PFN/active layer/MoO₃/Ag are increased from 4.97% to 8.54% and 5.92% to 8.74%, in contrast to those for the regular PVCs (<i>r-</i>PVCs) with devices configuration as ITO/PEDOT:PSS/active layer/Ca/Al under 100 mW/cm² AM 1.5 illumination. The optical modeling calculations and X-ray photoelectron spectroscopy (XPS) investigations reveal that the <i>r-</i>PVCs and <i>i-</i>PVCs from the copolymers exhibit similar light harvesting characteristics, and the enhancements of the PCEs of the <i>i-</i>PVCs from the copolymers are mainly contributed to the favorable vertical phase separation as the strongly polymer-enriched top surface layers and slightly PC₇₁BM (phenyl-C₇₁-butyric acid methyl ester)-enriched bottom surface layers are correspondingly connected to the anodes and cathodes of the <i>i-</i>PVCs, while they are opposite in the <i>r-</i>PVCs. As we known, it is the first time to experimentally verify that the <i>i-</i>PVCs with alcohol-soluble conjugated polymers cathode modification layers enjoy favorable vertical phase separation