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: PLoS Biology

Article Title: Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1

doi: 10.1371/journal.pbio.1000612

Figure Lengend Snippet: (A) Confocal micrograph showing mixed cytosolic and mitochondrial localization of GFP-Drp1 in PC12 cells (mito, MitoTracker Deep Red). (B–D) FRAP analysis in PC12 cells shows opposite effects of PKA activation and AKAP1 knockdown on GFP-Drp1 dynamics. PC12 cells co-expressing GFP-Drp1 and either AKAP1-directed or control shRNA were treated ± forskolin/rolipram (25/1 µM, 1–3 h) and Drp1 turnover was measured by bleaching mitochondrial GFP-Drp1 in a 5×5 µm square and monitoring fluorescence recovery at 5 s intervals. (B) shows frames from representative cells (control, forsk/roli: control shRNA ± forskolin/rolipram; shAKAP1: AKAP1 shRNA #1), (C) shows averaged fluorescence recovery curves, and (D) plots Drp1 turnover as the ratio of mobile fraction (mFx) and 50% recovery time (t 1/2 ) derived from biexponential fits ( R 2 ∼0.99) of individual recovery curves (means ± s.e.m. of 8–10 cells for each condition from a representative experiment). (E–F) Subcellular fractionation of Drp1. COS cells co-expressing GFP-Drp1 with either outer mitochondrial (om) PKA (+) or omGFP (−) were permeabilized with digitonin (500 µg/ml) and fractionated into a cytosolic (cyto) and a heavy membrane fraction containing mitochondria (mito). Fractions were immunoblotted for total Drp1, phospho-Ser PKA Drp1 (pDrp1), and the mitochondrial marker TOM40 and analyzed by densitometry (E, representative blot; F, summary showing means ± s.e.m. of 6 independent experiments).

Article Snippet: The following antibodies were used: rabbit anti-GFP (ab290, Abcam), mouse IgG 1 anti-MTCO2 (cytochrome oxidase subunit II, Neomarkers), rabbit anti-ERK (Santa Cruz), mouse anti-phospho-Ser PKA Drp1 , rabbit anti-phospho-Ser CDK Drp1 (Ser616, Cell Signaling), rabbit anti-LacZ and mouse IgG 2a anti-V5 epitope tag (Invitrogen), mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank, Iowa City), and mouse anti-MAP2B (BD Transduction Laboratories).

Techniques: Activation Assay, Knockdown, Expressing, Control, shRNA, Fluorescence, Derivative Assay, Fractionation, Membrane, Marker

Journal: PLoS Biology

Article Title: Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1

doi: 10.1371/journal.pbio.1000612

Figure Lengend Snippet: (A, B) PC12 cells expressing mitochondrial GFP (green) and either wild-type or S PKA A-mutant Drp1 instead of endogenous Drp1 were treated ± forskolin/rolipram (25/2 µM, 3 h), fixed, and epifluorescence micrographs (representatives in A) were subjected to digital morphometry (means ± s.e.m. of ∼300 cells per condition from a representative experiment). (C–D) HeLa cells co-expressing the indicated constructs (om, outer mitochondrial) were fixed and processed for immunofluorescence for mitochondrial cytochrome oxidase II (mito, red) and GFP (green). Shown are representative images (C) and mitochondrial morphology analysis (D, means ± s.e.m. of ∼200–300 cells per condition from a representative experiment). (E–G) HeLa cells expressing WT or S PKA D-mutant GFP-Drp1 were incubated for up to 4 h with the PKA inhibitor H89 (20 µM) and analyzed by quantitative immunoblotting for phosphorylated (pDrp1, Ser PKA , and Ser CDK ) and total Drp1 (E) or by immunofluorescence for mitochondrial morphology (F, representative image; G, means ± s.e.m. of ∼200 cells/condition from a representative experiment). For immunoblot analysis only (E), trace amounts (5% plasmid) of PKA catalytic subunit were cotransfected to increase the signal strength with the phospho-Ser PKA Drp1 antibody.

Article Snippet: The following antibodies were used: rabbit anti-GFP (ab290, Abcam), mouse IgG 1 anti-MTCO2 (cytochrome oxidase subunit II, Neomarkers), rabbit anti-ERK (Santa Cruz), mouse anti-phospho-Ser PKA Drp1 , rabbit anti-phospho-Ser CDK Drp1 (Ser616, Cell Signaling), rabbit anti-LacZ and mouse IgG 2a anti-V5 epitope tag (Invitrogen), mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank, Iowa City), and mouse anti-MAP2B (BD Transduction Laboratories).

Techniques: Expressing, Mutagenesis, Construct, Immunofluorescence, Incubation, Western Blot, Plasmid Preparation

Journal: PLoS Biology

Article Title: Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1

doi: 10.1371/journal.pbio.1000612

Figure Lengend Snippet: (A, B) COS cells co-expressing GFP-Drp1 and either empty vector, wild-type, or ΔPKA-mutant GFP-AKAP1 1–524 were stimulated for 45 min with increasing concentrations of forskolin/rolipram (rolipram at 1/30 th of the indicated forskolin concentrations), and total cell lysates were probed for phospho-Ser PKA Drp1 and GFP (detecting Drp1 and AKAP1) on the same blot using a dual-channel infrared imager. Drp1 phosphorylation was quantified as the ratio of phospho- to total Drp1 signals normalized to the highest value and is shown as part of the representative blots (A) and the summary graph (B, means ± s.e.m. of 6 independent experiments). (C, D) HeLa cells co-expressing the indicated constructs (ΔPKA = I310P, L316P-mutant) were treated with forskolin/rolipram (25/2 µM, 3 h) and processed for immunofluorescence for mitochondrial cytochrome oxidase II (mito, red) and V5-tagged AKAP1 (green). Shown are representative images (C) and mitochondrial morphology analysis (D, mean ± s.e.m. of ∼200–300 cells per condition from a representative experiment). (E, F) PC12 cells were cotransfected with GFP-Drp1/shRNA expression plasmids and either AKAP1 cDNAs or shRNAs. Cultures were treated 48 h posttransfection with 0.5 µM staurosporine for 24 h, fixed, and stained with Hoechst 33342. Representative images in (E) show overlays of cell contours (bright field), GFP-Drp1 (green), and Hoechst-stained nuclei (blue), with transfected cells displaying normal nuclear morphology and many untransfected cells having condensed, apoptotic nuclei (arrows). Apoptosis was quantified as the percentage of GFP-positive cells with condensed or fragmented nuclei (E, means ± s.e.m. of 6–13 image fields with ∼300 transfected cells per condition from one experiment representative of three).

Article Snippet: The following antibodies were used: rabbit anti-GFP (ab290, Abcam), mouse IgG 1 anti-MTCO2 (cytochrome oxidase subunit II, Neomarkers), rabbit anti-ERK (Santa Cruz), mouse anti-phospho-Ser PKA Drp1 , rabbit anti-phospho-Ser CDK Drp1 (Ser616, Cell Signaling), rabbit anti-LacZ and mouse IgG 2a anti-V5 epitope tag (Invitrogen), mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank, Iowa City), and mouse anti-MAP2B (BD Transduction Laboratories).

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Phospho-proteomics, Construct, Immunofluorescence, shRNA, Staining, Transfection

Journal: PLoS Biology

Article Title: Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1

doi: 10.1371/journal.pbio.1000612

Figure Lengend Snippet: (A–C) HeLa cells co-expressing wild-type or S PKA A-mutant GFP-Drp1 and either wild-type or PKA-binding deficient (ΔPKA) AKAP1 were subjected to FRAP analysis. (A) Frames showing time lapse series of representative cells (green: GFP-Drp1, red: MitoTracker Deep Red) with enlargements of the 180 s frame demonstrating recovery of Drp1 into mitochondrial foci (arrows). (B) Average recovery curves of cells expressing wild-type GFP-Drp1 and either wild-type or mutant AKAP1, and (C) plots mitochondrial Drp1 turnover as the 50% recovery time calculated from biexponential fits of individual recovery curves ( R 2 ∼0.992; means ± s.e.m. of 5–9 cells each from a representative experiment). (D) COS cells cotransfected with Drp1 and either omGFP or omPKA were treated for 5 min with the indicated concentration of the reversible, membrane permeant crosslinker dithiobismaleimidoethane (DTME), and cleared cell lysates were subjected to ultracentrifugation to sediment Drp1 complexes.

Article Snippet: The following antibodies were used: rabbit anti-GFP (ab290, Abcam), mouse IgG 1 anti-MTCO2 (cytochrome oxidase subunit II, Neomarkers), rabbit anti-ERK (Santa Cruz), mouse anti-phospho-Ser PKA Drp1 , rabbit anti-phospho-Ser CDK Drp1 (Ser616, Cell Signaling), rabbit anti-LacZ and mouse IgG 2a anti-V5 epitope tag (Invitrogen), mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank, Iowa City), and mouse anti-MAP2B (BD Transduction Laboratories).

Techniques: Expressing, Mutagenesis, Binding Assay, Concentration Assay, Membrane

Journal: PLoS Biology

Article Title: Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1

doi: 10.1371/journal.pbio.1000612

Figure Lengend Snippet: (A) Drp1 domain diagram. (B–C) Endogenous Drp1 in HeLa cells was replaced by transfection of the indicated ratios of wild-type (WT) and T55D mutant GFP-Drp1 expression plasmids. Cells were fixed and analyzed for mitochondrial length and Drp1 colocalization with mitochondria using ImageJ software , . (B) Representative images showing punctate localization of GFP-Drp1 T55D (green) on mitochondria (cytochrome oxidase subunit II antibody). (C) Correlation between mitochondrial length and mitochondrial localization of Drp1 as a function of Drp1 T55D expression (means ± s.e.m. of ∼200 cells per condition from a representative experiment). Wild-type and T55D-mutant Drp1 expression levels are similar and scale with plasmid amounts (inset). (D, E) Turnover of GFP-Drp1 WT and T55D was analyzed by FRAP as in . (D) Frames showing representative time lapse series (green: GFP-Drp1, red: MitoTracker Deep Red, see ) of HeLa cells expressing 100% WT or T55D Drp1, with the last frame expanded to show recovery of WT Drp1 into mitochondrial foci (arrows). (E) Average recovery curves (left) and curve fit-derived turnover (right, ratio of mobile fraction [mFx] and 50% recovery time [t 1/2 ]) from cells expressing varying ratios of WT and T55D-mutant GFP-Drp1 (mean ± s.e.m. of 12–20 cells each from a representative experiment). (F) COS cells expressing GFP-Drp1 WT or T55D were incubated with DTME (5 min, 500 µM), and cleared cell lysates were subjected to ultracentrifugation to sediment Drp1 complexes (∼2-fold increase with the T55D mutation).

Article Snippet: The following antibodies were used: rabbit anti-GFP (ab290, Abcam), mouse IgG 1 anti-MTCO2 (cytochrome oxidase subunit II, Neomarkers), rabbit anti-ERK (Santa Cruz), mouse anti-phospho-Ser PKA Drp1 , rabbit anti-phospho-Ser CDK Drp1 (Ser616, Cell Signaling), rabbit anti-LacZ and mouse IgG 2a anti-V5 epitope tag (Invitrogen), mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank, Iowa City), and mouse anti-MAP2B (BD Transduction Laboratories).

Techniques: Transfection, Mutagenesis, Expressing, Software, Plasmid Preparation, Derivative Assay, Incubation

Journal: PLoS Biology

Article Title: Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1

doi: 10.1371/journal.pbio.1000612

Figure Lengend Snippet: (A, B) HeLa cells transfected with GFP-Drp1 (green), dsRed2/mito (COX8 matrix targeting sequence, red), ± PKA catalytic subunit were imaged for ≥1 h at 37°C, capturing images every 30 s. (A) Representative frames of time lapse series (see Video S3 ). Blue lines connect GFP-Drp1 punctae that could be tracked for at least 5 min; x symbols denote mitochondrial fission events. Note that GFP-Drp1 punctae often split with the fragmenting mitochondrion. (B) Cumulative frequency plot of Drp1 punctae lifetimes and average lifetimes (bar graph inset; means ± s.e.m. of 35–62 cells and 11,000 to 40,000 punctae per condition from two independent experiments). (C) Model of mitochondrial fusion by PKA/AKAP1. GTP-bound Drp1 translocates to mitochondria to assemble into oligomeric complexes. Drp1 assembly stimulates GTP hydrolysis, leading to mitochondrial fission and release of Drp1 into the cytosol to complete the cycle. OMM-anchored PKA/AKAP1 phosphorylates Drp1 at Ser PKA , stabilizing the GTP-bound state to promote growth of Drp1 complexes to a size that is incompatible with membrane scission. Protein phosphatases (PP), including calcineurin, dephosphorylate Drp1 Ser PKA to return Drp1 into its active, rapidly cycling state.

Article Snippet: The following antibodies were used: rabbit anti-GFP (ab290, Abcam), mouse IgG 1 anti-MTCO2 (cytochrome oxidase subunit II, Neomarkers), rabbit anti-ERK (Santa Cruz), mouse anti-phospho-Ser PKA Drp1 , rabbit anti-phospho-Ser CDK Drp1 (Ser616, Cell Signaling), rabbit anti-LacZ and mouse IgG 2a anti-V5 epitope tag (Invitrogen), mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank, Iowa City), and mouse anti-MAP2B (BD Transduction Laboratories).

Techniques: Transfection, Sequencing, Membrane