Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Coimmunoprecipitation of SlrP and human ERdj3. HeLa cells were transiently co-transfected with 4 μg of two derivatives of plasmid pCS2, one expressing SlrP-3×FLAG, and the other ERdj3–3×HA (lane 1), or transfected with the plasmid expressing ERdj3–3×HA only (lane 2). Nonidet P-40 lysates from 5 × 106 transfected cells were subjected to immunoprecipitation (IP, right panels) with anti-FLAG monoclonal antibodies and, after the stringent washings described under “Experimental Procedures,” resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and developed with monoclonal anti-FLAG for the upper part of the membrane, and monoclonal anti-HA for the lower part. Lysates from 5 × 105 transfected cells are shown in the left panels.

Article Snippet: For each experiment, a lysate from 5 × 10 6 HeLa cells cotransfected with plasmids pIZ1720 and pIZ1729, expressing SlrP-3×FLAG and ERdj3–3xHA, respectively, and a lysate from 5 × 10 6 HeLa cells transfected with plasmid pIZ1729 only, were incubated with 4 μl of normal mouse IgG (Santa Cruz Biotechnology) for 1 h at 4 °C, and with 20 μl of protein A/G plus-agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C.

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Bioprocessing, SDS Page, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Colocalization of SlrP and ERdj3 in HeLa cells. A, microscopic analysis. HeLa cells transiently transfected with plasmids expressing SlrP-3×FLAG and ERdj3–3×HA were observed in a laser-scanning confocal microscope after permeabilization and staining with Alexa Fluor® 488 labeled anti-HA (green, upper panel) and Cy3-conjugated anti-FLAG (red, center panel). Superposition of the two labelings is shown in the lower panel. Yellowish staining indicates colocalization of SlrP and ERdj3 in the endoplasmic reticulum. Scale bar, 10 μm. B, detection of SlrP in the endoplasmic reticulum of HeLa-transfected cells. Total lysate (left panels) and microsomal fraction (right panels) from 107 HeLa cells stably expressing SlrP-3×FLAG were submitted to 15% SDS-PAGE, transferred to nitrocellulose membrane, and then probed with anti-FLAG monoclonal antibody to detect SlrP. The filter was also incubated with anti-ERdj3 and anti-Trx polyclonal antibodies to detect endogenous ERdj3, and endogenous Trx (a cytosolic and partially nuclear protein), respectively, as a control of the purity of the microsomal fraction. C, comparison of the migration of SlrP present in cytosolic and microsomal fractions of transfected HeLa cells. A cytosolic fraction (106 cells) and a microsomal fraction (107 cells) obtained from HeLa cells stably expressing SlrP-3×FLAG were submitted to 10% SDS-PAGE, transferred to nitrocellulose membrane and then probed with anti-FLAG monoclonal antibody to detect SlrP.

Article Snippet: For each experiment, a lysate from 5 × 10 6 HeLa cells cotransfected with plasmids pIZ1720 and pIZ1729, expressing SlrP-3×FLAG and ERdj3–3xHA, respectively, and a lysate from 5 × 10 6 HeLa cells transfected with plasmid pIZ1729 only, were incubated with 4 μl of normal mouse IgG (Santa Cruz Biotechnology) for 1 h at 4 °C, and with 20 μl of protein A/G plus-agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C.

Techniques: Transfection, Expressing, Microscopy, Staining, Labeling, Stable Transfection, SDS Page, Membrane, Incubation, Control, Comparison, Migration

Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Effect of SlrP on ERdj3 function. A, binding of ERdj3 to BiP. 50 μg of GST-ERdj3–1 (amino acids 23–358) or GST-ERdj3–3 (amino acids 129–358) immobilized in glutathione-agarose beads were incubated with 50 μg of His6-BiP in the presence or absence of 1 mm ATP and 50 μg of GST-SlrP as indicated. After incubating at 4 °C for 2 h, the beads were washed several times with binding buffer, and bound proteins were released by boiling for 5 min in sample buffer and subjected to 10% SDS-PAGE. Proteins were detected by Coomassie Blue staining. One stained gel is shown together with a graphical representation (below) of means ± S.D., from two independent experiments, of the quantification of bands of His6-Bip. B, stimulation of BiP ATPase activity by ERdj3. ATPase assays were performed with either no additions (None), 1 μg of His6-BiP plus 2.5 μg of GST (BiP), 1 μg of His6-BiP plus 2.5 μg of GST-ERdj3–1 plus 2.5 μg of GST (BiP+ERdj3), or 1 μg of His6-BiP plus 2.5 μg of GST-ERdj3–1 plus 2.5 μg GST-SlrP (BiP+ERdj3+SlrP). The amount of released phosphate was determined after incubation at 25 °C for 75 min with a non-radioactive procedure (see “Experimental Procedures”). Values are the mean of three separate experiments with an error bar representing S.D. C, binding of ERdj3 to dTg. Lysates from HeLa cells transiently expressing ERdj3–3×HA were mixed with lysates from untransfected cells (UTC) or with lysates transfected with SlrP or SlrPCys546Ala (C546A), as indicated, and then incubated with dTg or native protein A immobilized on agarose beads. After several washes with Nonidet P-40 buffer, proteins bound to the beads (Bi) were released by boiling in sample buffer, separated by using 10% SDS-PAGE and immunoblotted with anti-HA monoclonal antibodies. Part of the initial flow-through (F) obtained after the incubations was also included. D, image quantification of the relative amount of ERdj3 bound to immobilized dTg. The results are the mean ± S.D. of three independent experiments. The highest level was set to 100 for each experiment.

Article Snippet: For each experiment, a lysate from 5 × 10 6 HeLa cells cotransfected with plasmids pIZ1720 and pIZ1729, expressing SlrP-3×FLAG and ERdj3–3xHA, respectively, and a lysate from 5 × 10 6 HeLa cells transfected with plasmid pIZ1729 only, were incubated with 4 μl of normal mouse IgG (Santa Cruz Biotechnology) for 1 h at 4 °C, and with 20 μl of protein A/G plus-agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C.

Techniques: Binding Assay, Incubation, SDS Page, Staining, Activity Assay, Expressing, Transfection, Bioprocessing