Auxin stimulates PTRE1 accumulation at the plasma membrane.(a) Western blot analysis showed that auxin treatment results in relatively more PTRE1 at the plasma membrane, but decreased levels in nucleus and cytoplasm. Arabidopsis seedlings expressing PTRE1-GFP were treated with NAA (1??M, 60 or 120?min) and then surface-exposed membrane proteins were analysed using PTRE1 antibody or plasma membrane marker H+-ATPase. Nucleus and cytoplasm fractions were prepared from Col seedlings and analysed by western blot using antibody against PTRE1, nuclear marker H3 or cytoplasm marker UGPase. The relative quantities of the proteins were calculated by using image pro plus and indicated. (b) A proposed model for how PTRE1 and TIR1 coordinate auxin responses regulating proteasome activity and Aux/IAA protein degradation. Under normal condition (with basal auxin levels), 26S proteasome activity is maintained by appropriate distribution of PTRE1 at the plasma membrane and in intracellular compartments. In response to auxin, auxin rapidly stimulates the association of TIR1 and Aux/IAA proteins resulting in degradation of Aux/IAAs (1). Later, auxin suppresses PTRE1 to inhibit proteasome activity (possibly through stimulating the accumulation of PTRE1 at plasma membrane, resulting in decreased intracellular and nuclear localization) and hence suppresses Aux/IAA protein degradation (2) to coordinate regulation of auxin responses, reflecting a mechanism for fine control of Aux/IAA homeostasis and auxin signalling.

Development of transgenic rice with reduced As accumulation in the grains using tissue?specific expression of OsABCC1, ??ECS and ScYCF1. (a) Analysis of the tissue?specific expression of the OsRCc3 promoter by immunostaining OsRCc3 promoter::GUS transgenic plants (RCc3p::GUS) using a GUS antibody. The internode section denoted by broken lines is magnified in the inset surrounded by yellow solid lines. Bar = 100 ?m. (b) Maps of vectors carrying RCc3p?A (RCc3 pro::OsABCC1?V5), RCc3p?AE (RCc3 pro::OsABCC1?V5, ZmUBI pro::??ECS) or RCc3p?AEY (RCc3 pro::OsABCC1?V5, ZmUBI pro::??ECS, RCc3 pro::ScYCF1). (c) Subcellular localization of OsABCC1?V5 in the roots of RCc3p?AEY plants using a sucrose density gradient analysis. Anti?V5, anti?OsABCC1, anti?V?ATPase (tonoplast marker), anti?H+?ATPase (plasma membrane marker) and Bip (ER marker) were used to assess protein abundance in the different compartments. The signal of OsABCC1?V5 corresponded to the signals of OsABCC1 and V?ATPase. (d) Arsenic accumulation in brown rice harvested from T3 transgenic plants transformed with RCc3p?A, RCc3p?AE, RCc3p?AEY, UBIp?A, UBIp?AE or UBIp?AEY. The As concentration was analysed in brown rice from plants grown in soil with a typical basal level of As. The values are means and standard errors (n = 5 plants). Different letters indicate significantly different means (Tukey's multiple comparison analysis, P ? 0.05).

Total membrane fractions of Chara internodal cells.(A) Proteins of membrane fractions (MF) were separated by SDS-PAGE (10%), stained with Coomassie Brilliant Blue (CBB) or blotted onto PVDF membranes for immunodetection of the plasma membrane H+ ATPase. 30 ?g protein per lane. Numbers on the left refer to molecular weight markers in kDa. Only the upper part of the PVDF membrane was used, the lower part was probed for immunodetection of low molecular weight proteins. (B) Immunodetection of selected organelle marker proteins for vacuoles (VHA-?, H+ PPase), ER (BiP2), plasma membrane and endosomal compartments (ARA6) or cytosol (tubulin, GRF 14-3-3). Proteins of the MFs were separated by preparative SDS-PAGE (12.75% or 10% for GRF and ARA6), plotted onto PVDF membranes cut into 3 mm strips and detected with the respective antibodies. 10 ?g protein per strip. Molecular weight markers are given in kDa. Arrow heads indicate the expected position of the respective protein.

Differences in charasome abundance and protein expression in alkaline and acidic regions of Chara cells.(A) Left image pair: FM1-43-labelled charasomes (green fluorescence) and chloroplasts (bright field image) at an acidic band. Right image pair: FM1-43-stained charasomes are absent from the alkaline band; the bright field images show the chloroplasts. An FM1-43-stained internodal cell was cut into acid and alkaline regions as described in Materials and Methods guided by pH banding pattern visualized by phenol red. Cell fragments were mounted in artificial fresh water and examined in the CLSM. Bar = 20 ?m (B) Proteins of membrane fractions (MF) and cytosolic fractions (CF) obtained from acidic (ac) and alkaline (alk) regions were separated by SDS-PAGE (10%) and stained with Comassie Brilliant Blue (CBB) or blotted onto PVDF membranes for immunodetection of the plasma membrane H+ ATPase using only the upper part of the membrane. 15 ?g protein was loaded per lane. Numbers on the left refer to molecular weight markers in kDa. (C) Immunodetection of selected higher and lower molecular weight proteins from the membrane fraction (MF) as indicated. 10 ?g protein was loaded per lane, 10% gel. PVDF membrane was cut into upper and lower halves at 50 kDa. Molecular weight markers given in kDa.

Grape berry flesh cell protoplast and vacuole preparations and quality check. (A) Isolated protoplasts. (B) Vacuole enrichment by Ficoll gradient centrifugation. (C) Crude vacuoles stained with Neutral Red. (D) Vacuoles in vacuole-enriched preparation stained with Neutral Red, solid arrow indicating vacuole, dashed arrow indicating tonoplast vesicle, inset indicating vacuoles with microdomains. (E) Relative quantity of P-ATPase between protoplast and vacuole isolations by western blotting analysis. (F) Relative quantity of V-ATPase between protoplast and vacuole isolations by western blotting analysis. Values were the average of three replicates ± SD.

Co-immunoprecipitation analysis of plasma membrane H+-ATPase and 14-3-3 proteins. WT and transgenic Arabidopsis plants expressing FLAG-14-3-3? were grown in control liquid medium containing 100 mM Glc/30 mM N, and 10-day-old seedlings were treated with the control (C) or the high C/low N-nutrient stress medium containing 200 mM Glc/0.3 mM N (S) for 30 min. Proteins were extracted and subjected to immunoprecipitation with anti-FLAG antibody beads, followed by immunoblotting with anti-plasma membrane H+-ATPase and anti-FLAG antibodies. Three independent experiments showed similar results.