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RavJ induces a molecular weight shift in actin by a putative TG activity. (A) Portion of RavJ migrated as a higher molecular weight forms when expressed in mammalian cells. HEK293T cells were transfected to express Flag‐RavJ, RavJ C101A , RavJ H138A , or the RavJ D170A mutants. Total cell lysates were immunoprecipitated with beads coated with an anti‐Flag antibody. Products were resolved by SDS‐PAGE and probed with an anti‐Flag antibody. Note that RavJ displayed as an upshifted band on the blot. (B) Tandem purification of the Flag‐RavJ‐His 6 upshifted band. HEK293T cells were transfected with Flag‐RavJ‐His 6 , and cell lysates were immunoprecipitated using Flag antibody‐conjugated beads. Proteins were then eluted from the beads with <t>3XFLAG</t> peptides and incubated with Ni 2+ ‐NTA beads. Products were separated by SDS‐PAGE and detected by silver staining. (C) RavJ forms a covalent complex with actin in mammalian cells. HEK293T cells expressing the indicated proteins were immunoprecipitated using Flag antibody‐conjugated beads and analyzed by SDS‐PAGE. Samples were probed with the anti‐Flag (left) and anti‐RavJ (right) antibody.
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RavJ induces a molecular weight shift in actin by a putative TG activity. (A) Portion of RavJ migrated as a higher molecular weight forms when expressed in mammalian cells. HEK293T cells were transfected to express Flag‐RavJ, RavJ C101A , RavJ H138A , or the RavJ D170A mutants. Total cell lysates were immunoprecipitated with beads coated with an anti‐Flag antibody. Products were resolved by SDS‐PAGE and probed with an anti‐Flag antibody. Note that RavJ displayed as an upshifted band on the blot. (B) Tandem purification of the Flag‐RavJ‐His 6 upshifted band. HEK293T cells were transfected with Flag‐RavJ‐His 6 , and cell lysates were immunoprecipitated using Flag antibody‐conjugated beads. Proteins were then eluted from the beads with <t>3XFLAG</t> peptides and incubated with Ni 2+ ‐NTA beads. Products were separated by SDS‐PAGE and detected by silver staining. (C) RavJ forms a covalent complex with actin in mammalian cells. HEK293T cells expressing the indicated proteins were immunoprecipitated using Flag antibody‐conjugated beads and analyzed by SDS‐PAGE. Samples were probed with the anti‐Flag (left) and anti‐RavJ (right) antibody.
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RavJ induces a molecular weight shift in actin by a putative TG activity. (A) Portion of RavJ migrated as a higher molecular weight forms when expressed in mammalian cells. HEK293T cells were transfected to express Flag‐RavJ, RavJ C101A , RavJ H138A , or the RavJ D170A mutants. Total cell lysates were immunoprecipitated with beads coated with an anti‐Flag antibody. Products were resolved by SDS‐PAGE and probed with an anti‐Flag antibody. Note that RavJ displayed as an upshifted band on the blot. (B) Tandem purification of the Flag‐RavJ‐His 6 upshifted band. HEK293T cells were transfected with Flag‐RavJ‐His 6 , and cell lysates were immunoprecipitated using Flag antibody‐conjugated beads. Proteins were then eluted from the beads with 3XFLAG peptides and incubated with Ni 2+ ‐NTA beads. Products were separated by SDS‐PAGE and detected by silver staining. (C) RavJ forms a covalent complex with actin in mammalian cells. HEK293T cells expressing the indicated proteins were immunoprecipitated using Flag antibody‐conjugated beads and analyzed by SDS‐PAGE. Samples were probed with the anti‐Flag (left) and anti‐RavJ (right) antibody.

Journal: mLife

Article Title: Legionella pneumophila modulates the host cytoskeleton by an effector of transglutaminase activity

doi: 10.1002/mlf2.70013

Figure Lengend Snippet: RavJ induces a molecular weight shift in actin by a putative TG activity. (A) Portion of RavJ migrated as a higher molecular weight forms when expressed in mammalian cells. HEK293T cells were transfected to express Flag‐RavJ, RavJ C101A , RavJ H138A , or the RavJ D170A mutants. Total cell lysates were immunoprecipitated with beads coated with an anti‐Flag antibody. Products were resolved by SDS‐PAGE and probed with an anti‐Flag antibody. Note that RavJ displayed as an upshifted band on the blot. (B) Tandem purification of the Flag‐RavJ‐His 6 upshifted band. HEK293T cells were transfected with Flag‐RavJ‐His 6 , and cell lysates were immunoprecipitated using Flag antibody‐conjugated beads. Proteins were then eluted from the beads with 3XFLAG peptides and incubated with Ni 2+ ‐NTA beads. Products were separated by SDS‐PAGE and detected by silver staining. (C) RavJ forms a covalent complex with actin in mammalian cells. HEK293T cells expressing the indicated proteins were immunoprecipitated using Flag antibody‐conjugated beads and analyzed by SDS‐PAGE. Samples were probed with the anti‐Flag (left) and anti‐RavJ (right) antibody.

Article Snippet: Proteins were eluted from the corresponding beads using 3XFLAG peptides or HA peptides (Thermo Fisher Scientific).

Techniques: Molecular Weight, Activity Assay, Transfection, Immunoprecipitation, SDS Page, Purification, Incubation, Silver Staining, Expressing