Journal: Microbiology

Article Title: Degradation of SsrA-tagged proteins in streptococci

doi: 10.1099/mic.0.000048

Figure Lengend Snippet: S. mutans strains used in this study

Article Snippet: The abundance of cellular enolase was chosen as an internal control and was probed by an anti -S. mutans enolase rabbit polyclonal antibody (Genscript; I. Biswas, unpublished).

Techniques:

Journal: Microbiology

Article Title: Degradation of SsrA-tagged proteins in streptococci

doi: 10.1099/mic.0.000048

Figure Lengend Snippet: ClpXP degrades SsrA-tagged GFP in S. mutans. (a) Western blot analysis of GFPssrA and ClpX in the WT or clpX mutant transformed with an empty vector pIB190 (mock), clpX (pIBJ74, ClpX) or clpX mutant (pIBJ75, ClpXEGW). The blot was probed with anti-GFP antibody for GFP detection or anti-His antibody for ClpX detection. (b) Western blot analysis of GFP with or without different C-terminal tags in the WT S. mutans strain. The expression of enolase served as an internal control [detected using anti-enolase (Eno) antibody]. Protein expression from the plasmid is denoted by (p).

Article Snippet: The abundance of cellular enolase was chosen as an internal control and was probed by an anti -S. mutans enolase rabbit polyclonal antibody (Genscript; I. Biswas, unpublished).

Techniques: Western Blot, Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Control

Journal: Microbiology

Article Title: Degradation of SsrA-tagged proteins in streptococci

doi: 10.1099/mic.0.000048

Figure Lengend Snippet: S. mutans ClpP is unable to degrade SsrA- or AVAA-tagged GFP in vitro. (a) In vitro degradation of GFP, GFPssrA or GFPavaa by (a) ClpXP or (b) ClpP was monitored by measuring green fluorescence in the reaction mixture. The relative value of the initial GFP fluorescence was set as 1.

Article Snippet: The abundance of cellular enolase was chosen as an internal control and was probed by an anti -S. mutans enolase rabbit polyclonal antibody (Genscript; I. Biswas, unpublished).

Techniques: In Vitro, Fluorescence

Journal: Microbiology

Article Title: Degradation of SsrA-tagged proteins in streptococci

doi: 10.1099/mic.0.000048

Figure Lengend Snippet: ClpE and ClpC contribute to the degradation of AVAA-tagged GFP in S. mutans. (a) Western blot analysis of GFPavaa in the WT, clpP mutant, clpX mutant, ΔclpXΔclpE double mutant and ΔclpXΔclpC double mutant. (b) Growth kinetics of the ΔclpP mutant, ΔclpX mutant and ΔclpXΔclpCΔclpE triple mutant are similar in the antibiotic-free medium. (c) Western blot analysis of GFPavaa degradation. Protein samples from the bacterial cells at exponential phase (E), late exponential phase (LE) or stationary phase (S) as indicated by arrows in (b). (d) Complementation of ClpE in the ΔclpXΔclpCΔclpE mutant. Western blot of GFPavaa in the ΔclpX mutant or ΔclpXΔclpCΔclpE mutant transformed with the vector pGhost9tr (mock) or pIBJ96 (ClpE). The presence of ClpE in the mutants and the complemented strains was probed by an anti-ClpE antibody. (e) Complementation of ClpC in the ΔclpXΔclpCΔclpE mutant. Western blot of GFPavaa in the ΔclpX or ΔclpXΔclpCΔclpE mutant transformed with the vector pGhost9tr (mock) and triple mutant transformed with pIBJ97 (ClpC). Endogenous enolase was used for normalization in all the Western blot analyses.

Article Snippet: The abundance of cellular enolase was chosen as an internal control and was probed by an anti -S. mutans enolase rabbit polyclonal antibody (Genscript; I. Biswas, unpublished).

Techniques: Western Blot, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: Microbiology

Article Title: Degradation of SsrA-tagged proteins in streptococci

doi: 10.1099/mic.0.000048

Figure Lengend Snippet: ClpCP- and ClpEP-mediated degradation is induced by heat stress. (a) Western blot analysis of GFPavaa in the ΔclpXΔclpC and ΔclpXΔclpCΔctsR mutants. Cultures at both exponential phase (E) and late exponential phase (LE) were investigated. (b) Kinetics of SsrA degradation during heat stress. Total proteins were extracted from S. mutans cells grown either at 37 or 44 °C at the indicated time points t. (c) Level of GFPavaa in cell extracts was determined by Western blot analysis using an anti-GFP antibody. Enolase was used as an internal control.

Article Snippet: The abundance of cellular enolase was chosen as an internal control and was probed by an anti -S. mutans enolase rabbit polyclonal antibody (Genscript; I. Biswas, unpublished).

Techniques: Western Blot, Control