5203t  (Cell Signaling Technology Inc)

Journal: Cell

Article Title: Proximity-specific ribosome profiling reveals the logic of localized mitochondrial translation

doi: 10.1016/j.cell.2025.08.002

Figure Lengend Snippet: (A) Scatter plot comparing localized translation score (log 2 enrichment) in monoclonal AKAP1 null cells vs. wild-type using mitochondrial-specific LOCL-TL RNA-seq. CDSes less than 300 aa are in red, while the ones longer than 300 aa are in blue. (B) Bar graph showing enrichment of transcripts at the OMM in wild-type or AKAP1 null cells expressing different Myc-tagged AKAP1 protein variants or Myc-tagged mCherry as a control (N=3). A schematic at the top illustrates different AKAP1 domains. Normalization details are in the . p -values were generated by comparison to endogenous genes in wild-type cells. (C) Bar graph of enrichment scores for transcripts binding to different Myc-tagged AKAP1 variants, detected by RIP-qPCR (N=3). p -values were generated by comparison to endogenous genes in cells expressing wild-type Myc-tagged AKAP1. (D) Bar graph of relative enrichment for various NDUFB9 reporters binding to wild-type Myc-tagged AKAP1, detected by RIP-qPCR (N=3). Values for each reporter are normalized to the positive NDUFB9 reporter (top). (E) Violin plots of the log 2 fold change of mitochondrial protein abundance in monoclonal AKAP1 null cells compared to wild type, measured by quantitative proteomics. Short CDSes (mRNA mediated) are mitochondrial genes that are locally translated and contain fewer than 200 aa, as shown in . (F) Violin plots showing the log 2 fold change of mitochondrial protein abundance in different pathways in monoclonal AKAP1 null cells compared to wild type, measured by quantitative proteomics. p -values were generated by comparison to all mitochondrial genes. Statistical significance was determined by two-tailed unpaired t-test. p -value > 0.05 (ns), p -value ≤ 0.05 (*), p -value ≤ 0.01 (**), p -value ≤ 0.001 (***), p -value ≤ 0.0001 (****). See also .

Article Snippet: The following antibodies were used: a mouse anti-HA antibody (1:5000, Roche 12CA5), a mouse anti-FLAG antibody (1:1000, Sigma Aldrich F1804), a rabbit AKAP1 antibody (1:1000, Cell Signaling Technology 5203T), a rabbit LARP4 antibody (1:1000, courtesy from Richard J. Maraia′s lab), and a mouse anti-β-actin antibody (1:1000, Abcam 8226).

Techniques: RNA Sequencing, Expressing, Control, Generated, Comparison, Binding Assay, Quantitative Proteomics, Two Tailed Test

Journal: Cell

Article Title: Proximity-specific ribosome profiling reveals the logic of localized mitochondrial translation

doi: 10.1016/j.cell.2025.08.002

Figure Lengend Snippet: (A) Comparison of localized translation score for locally translated human mitochondrial genes and their yeast homologs. Colors match . MsRP stands for mitochondrial-specific ribosome profiling. (B) Bar graphs showing the percentage of prokaryotic vs. eukaryotic origin genes within different groups. (C) Comparison of localized translation scores for conserved ETC homologs. Prokaryotic origin genes are highlighted as red; eukaryotic origin genes are in black or dark gray. MsRP stands for mitochondrial-specific ribosome profiling. p -value ≤ 0.05 (*), p -value ≤ 0.01 (**). (D) Schematic summary model for three distinct pathways of nuclear-encoded mitochondrial protein import. The majority of proteins are imported post-translationally (left). This is mediated by a bipartite signal involving an N-terminal MTS, which is inhibited by the region immediately downstream of the MTS, preventing protein import until translation is completed. This inhibition may be mediated by chaperones and/or cochaperones that are in close proximity to or associated with ribosomes , . By contrast, a subset of proteins undergoes localized translation via two distinct pathways: Cotranslational targeting to the OMM (center): Proteins longer than approximately 400 aa are cotranslationally inserted into the OMM. These are mostly matrix proteins of prokaryotic origin. Their localized translation is driven by a bipartite targeting signal, which includes a generic MTS and additional amino acids just prior to engagement (e.g., residues 100–250) that do not inhibit the MTS. Translation-independent mRNA targeting to the OMM (right): Transcripts encoding short coding sequences (approximately less than 200 aa) are targeted to the OMM in a translation-independent manner via intron splicing and UTRs. This process is uncoupled from protein import, which still requires a functional MTS. These short CDS proteins are enriched in ETC subunits and are typically of eukaryotic origin. AKAP1 promotes the localized translation of short CDS transcripts by specifically binding and recruiting the targeted mRNAs.

Article Snippet: The following antibodies were used: a mouse anti-HA antibody (1:5000, Roche 12CA5), a mouse anti-FLAG antibody (1:1000, Sigma Aldrich F1804), a rabbit AKAP1 antibody (1:1000, Cell Signaling Technology 5203T), a rabbit LARP4 antibody (1:1000, courtesy from Richard J. Maraia′s lab), and a mouse anti-β-actin antibody (1:1000, Abcam 8226).

Techniques: Comparison, Inhibition, Functional Assay, Binding Assay

Journal: Advanced Exercise and Health Science

Article Title: Exercise-Triggered Cardiac Remodeling: AKIP1 as a Novel Mediator of Physiological Hypertrophy

doi: 10.1016/j.aehs.2025.05.002

Figure Lengend Snippet: Fig. 1. Changes in cardiac function after exercise in mice. (A) Ultrasound and electrocardiogram (ECG) images of mice. (B) Indices of cardiac function in mice. Con group: control group; EX group: exercise group; shAAV-AKIP1 +EX group: interference with AKIP1 adeno-associated virus expression in vivo injection+exercise group. LVMI: left ventricular mass index; LVID-d: left ventricular end-diastolic internal diameter; LVID-s: left ventricular end-systolic internal diameter; LVPW-d: left ventricular posterior wall thickness-diastolic; LVPW-s: left ventricular posterior wall thickness-systolic; EF: ejection fraction; FS: left ventricular shortening fraction. * indicates p < 0.05 compared with con group; ** indicates p < 0.01 compared with con group, ## indicates p < 0.01 compared with EX group.

Article Snippet: The protocol for immunoblot analysis has been previously described.23 And the blot was incubated with primary antibodies such as GAPDH (Cat # 5174, Cell Signaling Technology Inc., MA, USA), AKIP1 (Cat # 5203, Cell Signaling Technology Inc., MA, USA).

Techniques: Control, Virus, Expressing, In Vivo, Injection

Journal: Advanced Exercise and Health Science

Article Title: Exercise-Triggered Cardiac Remodeling: AKIP1 as a Novel Mediator of Physiological Hypertrophy

doi: 10.1016/j.aehs.2025.05.002

Figure Lengend Snippet: Fig. 2. Evaluation of animal models of exercise-in duced cardiac hypertrophy. (A) Representative photo micrographs of mice myocardial tissue stained with hematoxylin and eosin (HE). (B) ratio of heart weight to tibial length (HW/TL) in mice. (C) Expression of mRNA for markers of cardiac hypertrophy in mice. Con group: control group; EX group: exercise group; shAAV-AKIP1 +EX group: interference with AKIP1 adeno-associated virus expression in vivo injection +exercise group. ANP: natriuretic peptide A; BNP: brain natriuretic peptide; α-actin: α-smooth muscle actin; SERCA-2α: sarcoplasmic reticulum/endoplasmic reticulum calcium ATPase 2α; α-MHC: α-myosin heavy chain; β-MHC: β-myosin heavy chain. * indicates p < 0.05 compared with con group; ** indicates p < 0.01 compared with con group, ## indicates p < 0.01 compared with EX group.

Article Snippet: The protocol for immunoblot analysis has been previously described.23 And the blot was incubated with primary antibodies such as GAPDH (Cat # 5174, Cell Signaling Technology Inc., MA, USA), AKIP1 (Cat # 5203, Cell Signaling Technology Inc., MA, USA).

Techniques: Staining, Expressing, Control, Virus, In Vivo, Injection

Journal: Advanced Exercise and Health Science

Article Title: Exercise-Triggered Cardiac Remodeling: AKIP1 as a Novel Mediator of Physiological Hypertrophy

doi: 10.1016/j.aehs.2025.05.002

Figure Lengend Snippet: Fig. 3. Expression of related gene in the model of exercise-induced cardiac hypertrophy. (A) The level of cardiac AKIP1 mRNA in mice. (B) The level of cardiac AKIP1 protein in mice. (C) The level of cardiac PI3K(p110α) mRNA in mice. (D) The level of cardiac mTORC1 mRNA in mice. Con group: control group; EX group: exercise group; shAAV-AKIP1 +EX group: interference with AKIP1 adeno-associated virus expression in vivo injection+exercise group. AKIP1: a kinase interacting protein 1; PI3K(p110α): the p110α isoform of phosphatidylinositol-3-kinase; mTORC1: mechanistic target of rapamycin complex 1. ** indicates p < 0.01 compared with con group, ## indicates p < 0.01 compared with EX group.

Article Snippet: The protocol for immunoblot analysis has been previously described.23 And the blot was incubated with primary antibodies such as GAPDH (Cat # 5174, Cell Signaling Technology Inc., MA, USA), AKIP1 (Cat # 5203, Cell Signaling Technology Inc., MA, USA).

Techniques: Expressing, Control, Virus, In Vivo, Injection