Anti-BiP | Lumenal-binding protein (rabbit antibody)

Analysis of protein fractions from different steps of nuclei isolation. The protein fractions obtained following the main steps of the procedure used for isolation of nuclei from leaves of apple are shown. Equal amounts of proteins (4 ?g) from each fraction were loaded on the gel. A) Lumenal-binding protein 2 (BiP2), B) histone H3 and C) plastocyanin (PC) were detected with specific antibodies by western blot analysis. D) Coomassie blue staining of the fractionated proteins separated by polyacrylamide gel electrophoresis. Lane 1: crude extract of proteins from homogenized leaf tissue; lane 2: resuspended pellet of the whole cell lysate obtained following treatment with Triton X-100 and centrifugation; lane 3: nuclei collected from the 60% Percoll layer (see nuclear fraction in Figure 1B); lane 4: the interface fraction of 60% Percoll and 2.5 M sucrose layers in the density gradient containing chloroplasts and unbroken cells (see 60% P/2.5 M S interface in Figure 1B); lane 5: sucrose layer of the density gradient (see 2.5 M sucrose layer in Figure 1B); lane 6: nuclei purified by centrifugation on a 35% Percoll cushion.

(A–D) Western blot analyses of S. lycopersicum midrib extracts. Three healthy (lanes 1–3) and three infected (lanes 4–6) plants were analyzed. Immunoreactive signals corresponding to actin and ER luminal binding protein (BiP) displayed the expected molecular weights of ?40 and ?78 kDa, respectively (A). Amount of loaded protein were checked by Ponceau-S staining (B). Quantification of immunoreactive luminescence signals was achieved by densitometric scanning of the respective actin (C) and BiP (D) bands. The mean of six replicates (±SD) was calculated for each sample. Actin T-value: –9.977; BiP T-value: 15.068. * denotes significant difference of the means (P-values ? 0.001).

Accumulation of BiP, Catalase, and ATP synthase (beta chain) by western blot and analysis of spots. a Image of a nitrocellulose membrane hybridized with Anti-BiP, Anti-Catalase, and Anti-ATP synthase (beta chain) of Arabidopsis (Normalization, see Additional file 6: Figure S4). b Relative accumulation determined from images of the nitrocellulose membrane, using the Gel Quant. Net 1.8.0 software. c Relative quantification of each spot corresponding to proteins analyzed by western blot. The average values of the each spot’s percentage volume in each gel replicate was used for graph construction

Biochemical confirmation of AMP targeting activity. Mitochondrial, whole cell and chloroplast fractions (1 ?g protein per well) isolated from Chlamydomonas strains in which Venus localization is driven by bacillocin 1580 (A) or magainin II (B), each fused to the RBCA cleavage site, were immunolabelled with antibodies raised against FLAG, an epitope tag carried C-terminally by the Venus reporter, and markers for different cellular compartments: Cytochrome Oxidase subunit IIb (COXIIb) and ATPsynthase subunit F1? for mitochondria (mt), Photosystem II Oxygen Evolving Enhancer 2 (OEE2) and Rubisco small subunit (RBCS) for chloroplasts (cp), ?-Tubulin and nucleic-acid binding protein 1 (NAB1) for the cytosol (cyt) and luminal binding protein (BiP) for the endoplasmic reticulum (ER). Isolated chloroplasts from the Bacillocin 1580 strain (C) and isolated mitochondria from the Magainin II strain (D) were subjected to a proteinase assay, where aliquots were treated with either 150 ?g mL?1 proteinase K and/or 1% Triton X-100, a membrane solubilizing detergent. Aliquots were subsequently immuno-labelled with antibodies against FLAG, chloroplast ATP synthase subunit CF1 ? and other organelle-markers described aside.

Biochemical confirmation of AMP targeting activity. Mitochondrial, whole cell and chloroplast fractions (1 ?g protein per well) isolated from Chlamydomonas strains in which Venus localization is driven by bacillocin 1580 (A) or magainin II (B), each fused to the RBCA cleavage site, were immunolabelled with antibodies raised against FLAG, an epitope tag carried C-terminally by the Venus reporter, and markers for different cellular compartments: Cytochrome Oxidase subunit IIb (COXIIb) and ATPsynthase subunit F1? for mitochondria (mt), Photosystem II Oxygen Evolving Enhancer 2 (OEE2) and Rubisco small subunit (RBCS) for chloroplasts (cp), ?-Tubulin and nucleic-acid binding protein 1 (NAB1) for the cytosol (cyt) and luminal binding protein (BiP) for the endoplasmic reticulum (ER). Isolated chloroplasts from the Bacillocin 1580 strain (C) and isolated mitochondria from the Magainin II strain (D) were subjected to a proteinase assay, where aliquots were treated with either 150 ?g mL?1 proteinase K and/or 1% Triton X-100, a membrane solubilizing detergent. Aliquots were subsequently immuno-labelled with antibodies against FLAG, chloroplast ATP synthase subunit CF1 ? and other organelle-markers described aside.