Structured Review

GE Healthcare glutathione sepharose matrix
HrcQ interacts with itself, HrcN, HrcL and the cytoplasmic domains of HrcU and HrcV. (A) GST pull-down assays with HrcN. GST and GST-HrcN were immobilized on glutathione <t>sepharose</t> and incubated with a bacterial lysate containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) GST pull-down assays with HrcL. GST and GST-HrcL were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. (C) GST-HrcQ interacts with HrcL and HrcN. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcL-c-Myc and HrcN-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (D) HrcQ interacts with the cytoplasmic domain of HrcU. GST, GST-HrcU and GST-HrcU 255-357 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. GST-HrcU is cleaved at the conserved NPTH motif. The signals detected by the GST-specific antibody therefore correspond to GST-HrcU and the N-terminal cleavage product [26] . (E) HrcQ interacts with the cytoplasmic domain of HrcV and with itself. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcV-c-Myc, HrcV 324-645 -c-Myc, HrcQ-c-Myc or HrpB1-c-Myc. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown.
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1) Product Images from "HrcQ Provides a Docking Site for Early and Late Type III Secretion Substrates from Xanthomonas"

Article Title: HrcQ Provides a Docking Site for Early and Late Type III Secretion Substrates from Xanthomonas

Journal: PLoS ONE

doi: 10.1371/journal.pone.0051063

HrcQ interacts with itself, HrcN, HrcL and the cytoplasmic domains of HrcU and HrcV. (A) GST pull-down assays with HrcN. GST and GST-HrcN were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) GST pull-down assays with HrcL. GST and GST-HrcL were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. (C) GST-HrcQ interacts with HrcL and HrcN. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcL-c-Myc and HrcN-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (D) HrcQ interacts with the cytoplasmic domain of HrcU. GST, GST-HrcU and GST-HrcU 255-357 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. GST-HrcU is cleaved at the conserved NPTH motif. The signals detected by the GST-specific antibody therefore correspond to GST-HrcU and the N-terminal cleavage product [26] . (E) HrcQ interacts with the cytoplasmic domain of HrcV and with itself. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcV-c-Myc, HrcV 324-645 -c-Myc, HrcQ-c-Myc or HrpB1-c-Myc. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown.
Figure Legend Snippet: HrcQ interacts with itself, HrcN, HrcL and the cytoplasmic domains of HrcU and HrcV. (A) GST pull-down assays with HrcN. GST and GST-HrcN were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) GST pull-down assays with HrcL. GST and GST-HrcL were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. (C) GST-HrcQ interacts with HrcL and HrcN. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcL-c-Myc and HrcN-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (D) HrcQ interacts with the cytoplasmic domain of HrcU. GST, GST-HrcU and GST-HrcU 255-357 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. GST-HrcU is cleaved at the conserved NPTH motif. The signals detected by the GST-specific antibody therefore correspond to GST-HrcU and the N-terminal cleavage product [26] . (E) HrcQ interacts with the cytoplasmic domain of HrcV and with itself. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcV-c-Myc, HrcV 324-645 -c-Myc, HrcQ-c-Myc or HrpB1-c-Myc. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown.

Techniques Used: Incubation

HrcQ provides a docking site for early and late T3S substrates. (A) HrcQ interacts with T3S substrates and the T3S4 protein HpaC. GST, GST-XopF1, GST-XopA, GST-HrpB2, GST-HpaB, GST-HpaC, GST-HrpE, GST-AvrBs3, GST-XopJ and GST-HpaA were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) HrcQ interacts with XopJ and HpaA. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing XopJ-c-Myc and HpaA-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (C) The N-terminal region of AvrBs3 is dispensable for the interaction with HrcQ. GST, GST-AvrBs3, GST-AvrBs3Δ2 and GST-AvrBs3 1-50 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A.
Figure Legend Snippet: HrcQ provides a docking site for early and late T3S substrates. (A) HrcQ interacts with T3S substrates and the T3S4 protein HpaC. GST, GST-XopF1, GST-XopA, GST-HrpB2, GST-HpaB, GST-HpaC, GST-HrpE, GST-AvrBs3, GST-XopJ and GST-HpaA were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) HrcQ interacts with XopJ and HpaA. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing XopJ-c-Myc and HpaA-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (C) The N-terminal region of AvrBs3 is dispensable for the interaction with HrcQ. GST, GST-AvrBs3, GST-AvrBs3Δ2 and GST-AvrBs3 1-50 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A.

Techniques Used: Incubation

2) Product Images from "Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU"

Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

Journal: Molecular Microbiology

doi: 10.1111/j.1365-2958.2010.07461.x

The NPTH motif of HrcU contributes to the interaction between HrcU C and HrpB2. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. The P265G exchange abolishes the interaction between HrcU C and HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. D. GST–HrcU P265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.
Figure Legend Snippet: The NPTH motif of HrcU contributes to the interaction between HrcU C and HrpB2. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. The P265G exchange abolishes the interaction between HrcU C and HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. D. GST–HrcU P265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

Techniques Used: Incubation, Binding Assay

The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC. A. GST–HrcU 255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU 255–357 and GST–HrcU 255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. B. HrcU Y318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcU Y318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.
Figure Legend Snippet: The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC. A. GST–HrcU 255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU 255–357 and GST–HrcU 255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. B. HrcU Y318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcU Y318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.

Techniques Used: Mutagenesis, Incubation

The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. Mutations within the NPTH motif abolish the interaction between HrcU C and HpaC. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU 255–357 , GST–HrcU N264A , GST–HrcU P265A and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.
Figure Legend Snippet: The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. Mutations within the NPTH motif abolish the interaction between HrcU C and HpaC. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU 255–357 , GST–HrcU N264A , GST–HrcU P265A and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

Techniques Used: Incubation

3) Product Images from "The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System"

Article Title: The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01226-13

Interaction studies with HrpB1 deletion derivatives. (A) Amino acids 11 to 20 of HrpB1 contribute to the interaction of HrpB1 with itself, HrpB2, HrpE, and HrcD. GST, GST-HrpB1, GST-HrpB2, GST-HrpE, and GST-HrcD were immobilized on glutathione-Sepharose
Figure Legend Snippet: Interaction studies with HrpB1 deletion derivatives. (A) Amino acids 11 to 20 of HrpB1 contribute to the interaction of HrpB1 with itself, HrpB2, HrpE, and HrcD. GST, GST-HrpB1, GST-HrpB2, GST-HrpE, and GST-HrcD were immobilized on glutathione-Sepharose

Techniques Used:

Interaction studies with HrpB1 and HrpB2. (A) GST pulldown assays with HrpB1, HrpB2, and HrcD. GST, GST-HrpB1, and GST-HrpB2 were immobilized on glutathione-Sepharose and incubated with bacterial lysates containing HrpB1–c-Myc or HrcD–c-Myc.
Figure Legend Snippet: Interaction studies with HrpB1 and HrpB2. (A) GST pulldown assays with HrpB1, HrpB2, and HrcD. GST, GST-HrpB1, and GST-HrpB2 were immobilized on glutathione-Sepharose and incubated with bacterial lysates containing HrpB1–c-Myc or HrcD–c-Myc.

Techniques Used: Incubation

Interaction studies with HrpB1 point mutant derivatives. (A) GST, GST-HrpB1, GST-HrpB2, GST-HrpE, GST-HrcD, and GST-HrcC were immobilized on glutathione-Sepharose and incubated with bacterial lysates containing HrpB1–c-Myc or point mutant derivatives
Figure Legend Snippet: Interaction studies with HrpB1 point mutant derivatives. (A) GST, GST-HrpB1, GST-HrpB2, GST-HrpE, GST-HrcD, and GST-HrcC were immobilized on glutathione-Sepharose and incubated with bacterial lysates containing HrpB1–c-Myc or point mutant derivatives

Techniques Used: Mutagenesis, Incubation

4) Product Images from "Interaction of Staufen1 with the 5? end of mRNA facilitates translation of these RNAs"

Article Title: Interaction of Staufen1 with the 5? end of mRNA facilitates translation of these RNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki794

Binding of Stau1 55 to the 5′ end increases translation of structure-repressed transcripts. (A) Schematic representation of 5′-structure-repressed transcripts. RNAs coding for the R luc reporter protein are shown with one copy of the SBS or two copies of the MS2-binding site (MS2bs) at the 5′ end. (B) HEK293T cells were co-transfected with plasmids expressing either R luc or SBS- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of Stau1 55 -HA 3 , a 100-fold repression of translation of the SBS- R luc RNA was observed as compared with translation of R luc RNA. Results are expressed as luciferase activity versus concentration of the Stau1 55 -HA 3 coding plasmid. To facilitate comparison, the luciferase activity in the absence of Stau1 55 -HA 3 was defined as 1. P ≤ 0.01, n = 3. Black bars, SBS- R luc RNA; hatched bars, R luc RNA. (C) HEK293T cells were co-transfected with plasmids expressing the SBS- R luc transcript and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (D) HEK293T cells were co-transfected with plasmids expressing the MS2bs- R luc transcript and different concentrations of plasmids coding for either MS2-Stau1 55 -HA 3 , MS2-HA or Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of MS2-Stau1 55 -HA 3 , a 100-fold repression of translation of the MS2bs- R luc RNA was observed as compared with translation of R luc RNA. To facilitate comparison, the luciferase activity in the absence of expressor plasmids was defined as 1, n = 3.
Figure Legend Snippet: Binding of Stau1 55 to the 5′ end increases translation of structure-repressed transcripts. (A) Schematic representation of 5′-structure-repressed transcripts. RNAs coding for the R luc reporter protein are shown with one copy of the SBS or two copies of the MS2-binding site (MS2bs) at the 5′ end. (B) HEK293T cells were co-transfected with plasmids expressing either R luc or SBS- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of Stau1 55 -HA 3 , a 100-fold repression of translation of the SBS- R luc RNA was observed as compared with translation of R luc RNA. Results are expressed as luciferase activity versus concentration of the Stau1 55 -HA 3 coding plasmid. To facilitate comparison, the luciferase activity in the absence of Stau1 55 -HA 3 was defined as 1. P ≤ 0.01, n = 3. Black bars, SBS- R luc RNA; hatched bars, R luc RNA. (C) HEK293T cells were co-transfected with plasmids expressing the SBS- R luc transcript and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (D) HEK293T cells were co-transfected with plasmids expressing the MS2bs- R luc transcript and different concentrations of plasmids coding for either MS2-Stau1 55 -HA 3 , MS2-HA or Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of MS2-Stau1 55 -HA 3 , a 100-fold repression of translation of the MS2bs- R luc RNA was observed as compared with translation of R luc RNA. To facilitate comparison, the luciferase activity in the absence of expressor plasmids was defined as 1, n = 3.

Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Concentration Assay, Isolation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Stau1 55 mediated translational up-regulation does not involved RNA modification. (A) TAR-CAT RNA was incubated in RRL in the presence of 400 nM of bacterially expressed and purified Stau1 55 Δ2-his 6 or BSA for increasing periods of time. TAR-CAT RNA was then reverse transcribed and PCR amplified for 14 cycles to stay in the non-saturated part of the amplification curve. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (right panel). (B) HEK293T cells were co-transfected with plasmids expressing either R luc or TAR- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (C) Bacterially expressed and column-purified Stau1 55 Δ2-his 6 (Stau) and La-his 6 (La) (left panel) were incubated with [ 32 P]labelled double-stranded RNA in the presence of different combinations of ribonucleotides (right panel). RNA was resolved on agarose gel and revealed by autoradiography. While La-his 6 displayed an helicase activity, Stau1 55 Δ2-his 6 was inactive in this assay.
Figure Legend Snippet: Stau1 55 mediated translational up-regulation does not involved RNA modification. (A) TAR-CAT RNA was incubated in RRL in the presence of 400 nM of bacterially expressed and purified Stau1 55 Δ2-his 6 or BSA for increasing periods of time. TAR-CAT RNA was then reverse transcribed and PCR amplified for 14 cycles to stay in the non-saturated part of the amplification curve. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (right panel). (B) HEK293T cells were co-transfected with plasmids expressing either R luc or TAR- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (C) Bacterially expressed and column-purified Stau1 55 Δ2-his 6 (Stau) and La-his 6 (La) (left panel) were incubated with [ 32 P]labelled double-stranded RNA in the presence of different combinations of ribonucleotides (right panel). RNA was resolved on agarose gel and revealed by autoradiography. While La-his 6 displayed an helicase activity, Stau1 55 Δ2-his 6 was inactive in this assay.

Techniques Used: Modification, Incubation, Purification, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Transfection, Expressing, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Autoradiography, Activity Assay

5) Product Images from "Functional Characterization of the Type III Secretion Substrate Specificity Switch Protein HpaC from Xanthomonas campestris pv. vesicatoria ▿ ▿ †"

Article Title: Functional Characterization of the Type III Secretion Substrate Specificity Switch Protein HpaC from Xanthomonas campestris pv. vesicatoria ▿ ▿ †

Journal: Infection and Immunity

doi: 10.1128/IAI.00180-11

Protein-protein interaction studies with HrcU C and HpaC deletion derivatives. GST and GST-HrcU 255–357 were immobilized on glutathione Sepharose and incubated with E. coli lysates containing HpaC-c-Myc (HpaC) or HpaC-c-Myc deletion derivatives,
Figure Legend Snippet: Protein-protein interaction studies with HrcU C and HpaC deletion derivatives. GST and GST-HrcU 255–357 were immobilized on glutathione Sepharose and incubated with E. coli lysates containing HpaC-c-Myc (HpaC) or HpaC-c-Myc deletion derivatives,

Techniques Used: Incubation

6) Product Images from "Dephosphorylation of survival motor neurons (SMN) by PPM1G/PP2C? governs Cajal body localization and stability of the SMN complex"

Article Title: Dephosphorylation of survival motor neurons (SMN) by PPM1G/PP2C? governs Cajal body localization and stability of the SMN complex

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200704163

Interaction of PPM1G and the SMN complex. (A) SMN complex was precipitated from HeLa cell extract with a monoclonal anti-SMN antibody (7B10), covalently linked to protein G–Sepharose (IP-SMN). An unrelated mouse serum, covalently linked to protein G–Sepharose, was used as control (IP control). Interacting proteins were eluted by boiling in 2× SDS sample buffer, separated by SDS-PAGE, and analyzed by silver staining. (B) Immunoprecipitations of SMN complex (lane 3) or controls (lane 2) were done as described in A and analyzed by immunoblotting using specific SMN or PPM1G antibodies, respectively. Lane 1 shows HeLa cell total extract. (C) Interaction of recombinant PPM1G with the SMN complex. Immunoprecipitated SMN complex (lanes 2 and 3) or controls (lanes 4 and 5) were incubated with 5 μg of 6× histidine-tagged, purified, recombinant PPM1Gwt (rec.PPM1Gwt; lanes 2 and 4) or catalytically inactive PPM1G (D496A, rec.PPM1Gmut; lanes 3 and 5). Bound proteins were eluted by boiling in 2× SDS sample buffer, resolved by SDS-PAGE, and analyzed by silver staining. (D) Samples generated as described in C were analyzed by immunoblotting using anti-histidine (top), anti-unrip (middle), or anti-SMN antibodies (bottom). Note that because of different gels and markers used in B–D, the apparent molecular mass of PPM1G is slightly different in these panels.
Figure Legend Snippet: Interaction of PPM1G and the SMN complex. (A) SMN complex was precipitated from HeLa cell extract with a monoclonal anti-SMN antibody (7B10), covalently linked to protein G–Sepharose (IP-SMN). An unrelated mouse serum, covalently linked to protein G–Sepharose, was used as control (IP control). Interacting proteins were eluted by boiling in 2× SDS sample buffer, separated by SDS-PAGE, and analyzed by silver staining. (B) Immunoprecipitations of SMN complex (lane 3) or controls (lane 2) were done as described in A and analyzed by immunoblotting using specific SMN or PPM1G antibodies, respectively. Lane 1 shows HeLa cell total extract. (C) Interaction of recombinant PPM1G with the SMN complex. Immunoprecipitated SMN complex (lanes 2 and 3) or controls (lanes 4 and 5) were incubated with 5 μg of 6× histidine-tagged, purified, recombinant PPM1Gwt (rec.PPM1Gwt; lanes 2 and 4) or catalytically inactive PPM1G (D496A, rec.PPM1Gmut; lanes 3 and 5). Bound proteins were eluted by boiling in 2× SDS sample buffer, resolved by SDS-PAGE, and analyzed by silver staining. (D) Samples generated as described in C were analyzed by immunoblotting using anti-histidine (top), anti-unrip (middle), or anti-SMN antibodies (bottom). Note that because of different gels and markers used in B–D, the apparent molecular mass of PPM1G is slightly different in these panels.

Techniques Used: SDS Page, Silver Staining, Recombinant, Immunoprecipitation, Incubation, Purification, Generated

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Centrifugation:

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Affinity Chromatography:

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Purification:

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Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression
Article Snippet: .. GST-M6CK protein was purified from expressing BL21 bacteria using glutathione Sepharose (GE Healthcare) as described ( ). .. Purification of PRMT5, WDR77, and ICLN proteins was as described ( , ).

Expressing:

Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression
Article Snippet: .. GST-M6CK protein was purified from expressing BL21 bacteria using glutathione Sepharose (GE Healthcare) as described ( ). .. Purification of PRMT5, WDR77, and ICLN proteins was as described ( , ).

Sonication:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
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Affinity Purification:

Article Title: The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System
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Recombinant:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
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    GE Healthcare glutathione sepharose matrix
    HrcQ interacts with itself, HrcN, HrcL and the cytoplasmic domains of HrcU and HrcV. (A) GST pull-down assays with HrcN. GST and GST-HrcN were immobilized on glutathione <t>sepharose</t> and incubated with a bacterial lysate containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) GST pull-down assays with HrcL. GST and GST-HrcL were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. (C) GST-HrcQ interacts with HrcL and HrcN. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcL-c-Myc and HrcN-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (D) HrcQ interacts with the cytoplasmic domain of HrcU. GST, GST-HrcU and GST-HrcU 255-357 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. GST-HrcU is cleaved at the conserved NPTH motif. The signals detected by the GST-specific antibody therefore correspond to GST-HrcU and the N-terminal cleavage product [26] . (E) HrcQ interacts with the cytoplasmic domain of HrcV and with itself. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcV-c-Myc, HrcV 324-645 -c-Myc, HrcQ-c-Myc or HrpB1-c-Myc. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown.
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    HrcQ interacts with itself, HrcN, HrcL and the cytoplasmic domains of HrcU and HrcV. (A) GST pull-down assays with HrcN. GST and GST-HrcN were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) GST pull-down assays with HrcL. GST and GST-HrcL were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. (C) GST-HrcQ interacts with HrcL and HrcN. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcL-c-Myc and HrcN-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (D) HrcQ interacts with the cytoplasmic domain of HrcU. GST, GST-HrcU and GST-HrcU 255-357 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. GST-HrcU is cleaved at the conserved NPTH motif. The signals detected by the GST-specific antibody therefore correspond to GST-HrcU and the N-terminal cleavage product [26] . (E) HrcQ interacts with the cytoplasmic domain of HrcV and with itself. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcV-c-Myc, HrcV 324-645 -c-Myc, HrcQ-c-Myc or HrpB1-c-Myc. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown.

    Journal: PLoS ONE

    Article Title: HrcQ Provides a Docking Site for Early and Late Type III Secretion Substrates from Xanthomonas

    doi: 10.1371/journal.pone.0051063

    Figure Lengend Snippet: HrcQ interacts with itself, HrcN, HrcL and the cytoplasmic domains of HrcU and HrcV. (A) GST pull-down assays with HrcN. GST and GST-HrcN were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) GST pull-down assays with HrcL. GST and GST-HrcL were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. (C) GST-HrcQ interacts with HrcL and HrcN. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcL-c-Myc and HrcN-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (D) HrcQ interacts with the cytoplasmic domain of HrcU. GST, GST-HrcU and GST-HrcU 255-357 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A. GST-HrcU is cleaved at the conserved NPTH motif. The signals detected by the GST-specific antibody therefore correspond to GST-HrcU and the N-terminal cleavage product [26] . (E) HrcQ interacts with the cytoplasmic domain of HrcV and with itself. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcV-c-Myc, HrcV 324-645 -c-Myc, HrcQ-c-Myc or HrpB1-c-Myc. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown.

    Article Snippet: Insoluble cell debris were removed by centrifugation, and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer’s instructions (GE Healthcare, Munich, Germany).

    Techniques: Incubation

    HrcQ provides a docking site for early and late T3S substrates. (A) HrcQ interacts with T3S substrates and the T3S4 protein HpaC. GST, GST-XopF1, GST-XopA, GST-HrpB2, GST-HpaB, GST-HpaC, GST-HrpE, GST-AvrBs3, GST-XopJ and GST-HpaA were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) HrcQ interacts with XopJ and HpaA. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing XopJ-c-Myc and HpaA-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (C) The N-terminal region of AvrBs3 is dispensable for the interaction with HrcQ. GST, GST-AvrBs3, GST-AvrBs3Δ2 and GST-AvrBs3 1-50 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A.

    Journal: PLoS ONE

    Article Title: HrcQ Provides a Docking Site for Early and Late Type III Secretion Substrates from Xanthomonas

    doi: 10.1371/journal.pone.0051063

    Figure Lengend Snippet: HrcQ provides a docking site for early and late T3S substrates. (A) HrcQ interacts with T3S substrates and the T3S4 protein HpaC. GST, GST-XopF1, GST-XopA, GST-HrpB2, GST-HpaB, GST-HpaC, GST-HrpE, GST-AvrBs3, GST-XopJ and GST-HpaA were immobilized on glutathione sepharose and incubated with bacterial lysates containing HrcQ-c-Myc. Total cell extracts (TE) and eluted proteins (eluates) were analyzed by immunoblotting using c-Myc epitope- and GST-specific antibodies. GST and GST fusion proteins are marked with asterisks, additional bands correspond to degradation products. (B) HrcQ interacts with XopJ and HpaA. GST and GST-HrcQ were immobilized on glutathione sepharose and incubated with bacterial lysates containing XopJ-c-Myc and HpaA-c-Myc, respectively. TE and eluates were analyzed as is described in panel A. One representative blot incubated with GST-specific antibodies is shown. (C) The N-terminal region of AvrBs3 is dispensable for the interaction with HrcQ. GST, GST-AvrBs3, GST-AvrBs3Δ2 and GST-AvrBs3 1-50 were immobilized on glutathione sepharose and incubated with a bacterial lysate containing HrcQ-c-Myc. TE and eluates were analyzed as is described in panel A.

    Article Snippet: Insoluble cell debris were removed by centrifugation, and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer’s instructions (GE Healthcare, Munich, Germany).

    Techniques: Incubation

    The NPTH motif of HrcU contributes to the interaction between HrcU C and HrpB2. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. The P265G exchange abolishes the interaction between HrcU C and HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. D. GST–HrcU P265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

    Journal: Molecular Microbiology

    Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

    doi: 10.1111/j.1365-2958.2010.07461.x

    Figure Lengend Snippet: The NPTH motif of HrcU contributes to the interaction between HrcU C and HrpB2. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. The P265G exchange abolishes the interaction between HrcU C and HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. D. GST–HrcU P265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

    Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Incubation, Binding Assay

    The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC. A. GST–HrcU 255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU 255–357 and GST–HrcU 255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. B. HrcU Y318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcU Y318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.

    Journal: Molecular Microbiology

    Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

    doi: 10.1111/j.1365-2958.2010.07461.x

    Figure Lengend Snippet: The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC. A. GST–HrcU 255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU 255–357 and GST–HrcU 255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. B. HrcU Y318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcU Y318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.

    Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Mutagenesis, Incubation

    The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. Mutations within the NPTH motif abolish the interaction between HrcU C and HpaC. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU 255–357 , GST–HrcU N264A , GST–HrcU P265A and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

    Journal: Molecular Microbiology

    Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

    doi: 10.1111/j.1365-2958.2010.07461.x

    Figure Lengend Snippet: The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. Mutations within the NPTH motif abolish the interaction between HrcU C and HpaC. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU 255–357 , GST–HrcU N264A , GST–HrcU P265A and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

    Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Incubation

    Interaction studies with HrpB1 deletion derivatives. (A) Amino acids 11 to 20 of HrpB1 contribute to the interaction of HrpB1 with itself, HrpB2, HrpE, and HrcD. GST, GST-HrpB1, GST-HrpB2, GST-HrpE, and GST-HrcD were immobilized on glutathione-Sepharose

    Journal: Applied and Environmental Microbiology

    Article Title: The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System

    doi: 10.1128/AEM.01226-13

    Figure Lengend Snippet: Interaction studies with HrpB1 deletion derivatives. (A) Amino acids 11 to 20 of HrpB1 contribute to the interaction of HrpB1 with itself, HrpB2, HrpE, and HrcD. GST, GST-HrpB1, GST-HrpB2, GST-HrpE, and GST-HrcD were immobilized on glutathione-Sepharose

    Article Snippet: After centrifugation, comparable amounts of soluble GST and GST fusion proteins were immobilized on a glutathione-Sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques:

    Interaction studies with HrpB1 and HrpB2. (A) GST pulldown assays with HrpB1, HrpB2, and HrcD. GST, GST-HrpB1, and GST-HrpB2 were immobilized on glutathione-Sepharose and incubated with bacterial lysates containing HrpB1–c-Myc or HrcD–c-Myc.

    Journal: Applied and Environmental Microbiology

    Article Title: The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System

    doi: 10.1128/AEM.01226-13

    Figure Lengend Snippet: Interaction studies with HrpB1 and HrpB2. (A) GST pulldown assays with HrpB1, HrpB2, and HrcD. GST, GST-HrpB1, and GST-HrpB2 were immobilized on glutathione-Sepharose and incubated with bacterial lysates containing HrpB1–c-Myc or HrcD–c-Myc.

    Article Snippet: After centrifugation, comparable amounts of soluble GST and GST fusion proteins were immobilized on a glutathione-Sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Incubation

    Interaction studies with HrpB1 point mutant derivatives. (A) GST, GST-HrpB1, GST-HrpB2, GST-HrpE, GST-HrcD, and GST-HrcC were immobilized on glutathione-Sepharose and incubated with bacterial lysates containing HrpB1–c-Myc or point mutant derivatives

    Journal: Applied and Environmental Microbiology

    Article Title: The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System

    doi: 10.1128/AEM.01226-13

    Figure Lengend Snippet: Interaction studies with HrpB1 point mutant derivatives. (A) GST, GST-HrpB1, GST-HrpB2, GST-HrpE, GST-HrcD, and GST-HrcC were immobilized on glutathione-Sepharose and incubated with bacterial lysates containing HrpB1–c-Myc or point mutant derivatives

    Article Snippet: After centrifugation, comparable amounts of soluble GST and GST fusion proteins were immobilized on a glutathione-Sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Mutagenesis, Incubation

    Protein-protein interaction studies with HrcU C and HpaC deletion derivatives. GST and GST-HrcU 255–357 were immobilized on glutathione Sepharose and incubated with E. coli lysates containing HpaC-c-Myc (HpaC) or HpaC-c-Myc deletion derivatives,

    Journal: Infection and Immunity

    Article Title: Functional Characterization of the Type III Secretion Substrate Specificity Switch Protein HpaC from Xanthomonas campestris pv. vesicatoria ▿ ▿ †

    doi: 10.1128/IAI.00180-11

    Figure Lengend Snippet: Protein-protein interaction studies with HrcU C and HpaC deletion derivatives. GST and GST-HrcU 255–357 were immobilized on glutathione Sepharose and incubated with E. coli lysates containing HpaC-c-Myc (HpaC) or HpaC-c-Myc deletion derivatives,

    Article Snippet: Insoluble cell debris was removed by centrifugation, and soluble GST and GST fusion proteins were immobilized on a glutathione Sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Incubation