rabbit phospho cdk substrate motif  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

Figure Lengend Snippet: (A) WT and GSDMD knockout (GSDMD −/− ) iBMDMs were primed with LPS and stimulated with 10 μM nigericin over a time course of 45 min. Cellular lysates were examined by western blot for changes in phosphorylation of the ERK substrate motif PXSP. (B) Direct ERK targets as identified by Ünal et al. were probed for enriched molecular functions using WebGestalt. (C) A Venn diagram describing the overlap between RNA-seq differentially expressed genes (from ) and the RNA binding subset of ERK substrates. (D) A plot of the density of phosphorylation sites per kilodalton of protein mass in the human proteome, based on the PhosphoSitePlus database. TTP has a reported 1.35 phosphorylation sites per kDa. (E) A heatmap showing relative expression levels of TTP target genes in THP-1 monocytes after 0, 2, or 4 h of nigericin stimulation. (F) Western blots are representative of at least three independent experiments.

Article Snippet: Rabbit monoclonal anti-phospho-MAPK substrate (PXSP) , Cell Signaling Technology , Cat# 2325; RRID:AB_331820.

Techniques: Knock-Out, Western Blot, Phospho-proteomics, RNA Sequencing, RNA Binding Assay, Expressing

Journal: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

Figure Lengend Snippet: (A) WT and GSDMD knockout (GSDMD −/− ) iBMDMs were primed with LPS and stimulated with 10 μM nigericin over a time course of 45 min. Cellular lysates were examined by western blot for changes in electrophoretic mobility of TTP. (B) TTP knockout iBMDMs were generated on a WT and GSDMD −/− background using CRISPR-Cas9 technology. Individual clones were assessed for knockout via sequencing and western blot. Knockout clones were then pooled and assessed via western blot (top). TTP −/− cells were then reconstituted with empty vector (EV), WT TTP, TTP S220A, or TTP 5S to A (bottom). (C) TTP −/− iBMDMs were reconstituted with empty vector or myc-TTP. After a 45-min nigericin time course, myc-TTP was immunoprecipitated from whole cell lysate and assayed via western blot for phosphorylation of the ERK substrate motif PXSP. (D) A schematic of TTP outlining its functional domains and highlighting its two PXSP sites. Sequences surrounding the PXSP sites are given for mouse, human, and rat TTP, as well as drosophila Tis11 (a TTP analog). All numbering refers to the mouse protein. NES, nuclear export signal. (E) HEK293T cells were transiently transfected with ERK1, constitutively active MEK1, and WT or phosphosite mutant TTP. TTP was immunoprecipitated from whole cell lysate and the pulldown was assayed for phospho-PXSP motifs via western blot. (F) TTP −/− iBMDMs were reconstituted with WT and S220A myc-TTP. After a 45-min nigericin time course, myc-TTP was immunoprecipitated from whole cell lysate and assayed via western blot for phosphorylation of the ERK substrate motif PXSP. Western blots are representative of at least three independent experiments.

Article Snippet: Rabbit monoclonal anti-phospho-MAPK substrate (PXSP) , Cell Signaling Technology , Cat# 2325; RRID:AB_331820.

Techniques: Knock-Out, Western Blot, Generated, CRISPR, Clone Assay, Sequencing, Plasmid Preparation, Immunoprecipitation, Phospho-proteomics, Functional Assay, Transfection, Mutagenesis

Journal: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho-MAPK substrate (PXSP) , Cell Signaling Technology , Cat# 2325; RRID:AB_331820.

Techniques: Recombinant, CyQUANT Assay, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

Figure Lengend Snippet: (A) WT and GSDMD knockout (GSDMD −/− ) iBMDMs were primed with LPS and stimulated with 10 μM nigericin over a time course of 45 min. Cellular lysates were examined by western blot for changes in phosphorylation of the ERK substrate motif PXSP. (B) Direct ERK targets as identified by Ünal et al. were probed for enriched molecular functions using WebGestalt. (C) A Venn diagram describing the overlap between RNA-seq differentially expressed genes (from ) and the RNA binding subset of ERK substrates. (D) A plot of the density of phosphorylation sites per kilodalton of protein mass in the human proteome, based on the PhosphoSitePlus database. TTP has a reported 1.35 phosphorylation sites per kDa. (E) A heatmap showing relative expression levels of TTP target genes in THP-1 monocytes after 0, 2, or 4 h of nigericin stimulation. (F) Western blots are representative of at least three independent experiments.

Article Snippet: Antibodies used were phospho-ERK1/2 (Cell Signaling #9106), ERK1/2 (Cell Signaling #4695), TTP (Cell Signaling #71632), phospho-MEK1/2 (Cell Signaling #9154), MEK1/2 (Cell Signaling #8727), myc-tag (Cell Signaling #2278), phospho-PXSP (Cell Signaling #2325), GSDMD (Abcam #ab209845), HRP-linked anti-rabbit IgG (Cell Signaling #7074), and HRP-linked anti-mouse IgG (Cell Signaling #7076).

Techniques: Knock-Out, Western Blot, Phospho-proteomics, RNA Sequencing, RNA Binding Assay, Expressing

Journal: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

Figure Lengend Snippet: (A) WT and GSDMD knockout (GSDMD −/− ) iBMDMs were primed with LPS and stimulated with 10 μM nigericin over a time course of 45 min. Cellular lysates were examined by western blot for changes in electrophoretic mobility of TTP. (B) TTP knockout iBMDMs were generated on a WT and GSDMD −/− background using CRISPR-Cas9 technology. Individual clones were assessed for knockout via sequencing and western blot. Knockout clones were then pooled and assessed via western blot (top). TTP −/− cells were then reconstituted with empty vector (EV), WT TTP, TTP S220A, or TTP 5S to A (bottom). (C) TTP −/− iBMDMs were reconstituted with empty vector or myc-TTP. After a 45-min nigericin time course, myc-TTP was immunoprecipitated from whole cell lysate and assayed via western blot for phosphorylation of the ERK substrate motif PXSP. (D) A schematic of TTP outlining its functional domains and highlighting its two PXSP sites. Sequences surrounding the PXSP sites are given for mouse, human, and rat TTP, as well as drosophila Tis11 (a TTP analog). All numbering refers to the mouse protein. NES, nuclear export signal. (E) HEK293T cells were transiently transfected with ERK1, constitutively active MEK1, and WT or phosphosite mutant TTP. TTP was immunoprecipitated from whole cell lysate and the pulldown was assayed for phospho-PXSP motifs via western blot. (F) TTP −/− iBMDMs were reconstituted with WT and S220A myc-TTP. After a 45-min nigericin time course, myc-TTP was immunoprecipitated from whole cell lysate and assayed via western blot for phosphorylation of the ERK substrate motif PXSP. Western blots are representative of at least three independent experiments.

Article Snippet: Antibodies used were phospho-ERK1/2 (Cell Signaling #9106), ERK1/2 (Cell Signaling #4695), TTP (Cell Signaling #71632), phospho-MEK1/2 (Cell Signaling #9154), MEK1/2 (Cell Signaling #8727), myc-tag (Cell Signaling #2278), phospho-PXSP (Cell Signaling #2325), GSDMD (Abcam #ab209845), HRP-linked anti-rabbit IgG (Cell Signaling #7074), and HRP-linked anti-mouse IgG (Cell Signaling #7076).

Techniques: Knock-Out, Western Blot, Generated, CRISPR, Clone Assay, Sequencing, Plasmid Preparation, Immunoprecipitation, Phospho-proteomics, Functional Assay, Transfection, Mutagenesis

Journal: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

Figure Lengend Snippet:

Article Snippet: Antibodies used were phospho-ERK1/2 (Cell Signaling #9106), ERK1/2 (Cell Signaling #4695), TTP (Cell Signaling #71632), phospho-MEK1/2 (Cell Signaling #9154), MEK1/2 (Cell Signaling #8727), myc-tag (Cell Signaling #2278), phospho-PXSP (Cell Signaling #2325), GSDMD (Abcam #ab209845), HRP-linked anti-rabbit IgG (Cell Signaling #7074), and HRP-linked anti-mouse IgG (Cell Signaling #7076).

Techniques: Recombinant, CyQUANT Assay, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Software