Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: TRIM40 interacts directly with PKN2. (A) Schematic diagram of the quantitative proteomic screening workflow for identifying TRIM40‐interacting proteins. (B) Tandem mass spectrum of representative peptide fragments from PKN2. (C) Amino acid sequence information of the identified PKN2 peptides. (D, E) Co‐IP assays using anti‐Flag antibody in NRVMs (D) and HEK‐293T cells (E) transfected with Flag‐tagged TRIM40, followed by immunoblotting to detect PKN2 association. IgG served as a NC for Co‐IP (n = 3). (F) Endogenous PKN2 binding was detected by immunoblotting after Co‐IP with anti‐TRIM40 antibody from mouse heart tissue lysates. IgG was used as a control (n = 6). (G) Schematic representation of the domain deletion mutants of PKN2. (H) HEK‐293T cells were co‐transfected with HA‐tagged full‐length PKN2 or its deletion mutants together with Flag‐TRIM40. Immunoprecipitation was performed using anti‐HA antibody, followed by immunoblotting to detect Flag‐TRIM40 binding (n = 3). (I) Schematic diagrams of TRIM40 domain deletion mutants and its catalytically inactive mutant (C29S). (J) HEK‐293T cells were co‐transfected with Flag‐tagged full‐length TRIM40 or its mutants together with HA‐PKN2. Immunoprecipitation with anti‐Flag antibody was used to assess HA‐PKN2 binding (n = 3). (K) Ubiquitination assay of PKN2 in HEK‐293T cells co‐expressing Myc‐Ub, HA‐PKN2, and the catalytically inactive mutant Flag‐TRIM40‐C29S. HA immunoprecipitates were analyzed by immunoblotting to detect PKN2 ubiquitination. (n = 3) (L) Confocal microscopy images showing the effects of different TRIM40 variants (full‐length, deletion mutants, and C29S mutant) on F‐actin cytoskeleton organization in NRVMs. Cells were stained with rhodamine‐conjugated phalloidin (red, labeling F‐actin) and anti‐TRIM40 antibody (green, indicating transfected TRIM40 variants), with nuclei counterstained by DAPI (blue). Scale bar = 50 µm (n = 3). (M) Structural basis of the TRIM40‐PKN2 interaction.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Sequencing, Co-Immunoprecipitation Assay, Transfection, Western Blot, Binding Assay, Control, Immunoprecipitation, Mutagenesis, Ubiquitin Proteomics, Expressing, Confocal Microscopy, Staining, Labeling

Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: TRIM40 Positively Regulates the Phosphorylation of PKN2 by Mediating Its K63‐Linked Ubiquitination. (A) HEK‐293T cells were transfected with HA‐PKN2, Myc‐Ub, and Flag‐TRIM40, and treated with 10 µ m MG132 for 6 h before harvesting. The ubiquitination level of PKN2 was detected by immunoblotting using an anti‐HA antibody (Control = IgG) (n = 3). (B) HEK‐293T cells were co‐transfected with HA‐PKN2, Flag‐TRIM40, and various types of Myc‐Ub (including WT, K6‐, K11‐, K27‐, K29‐, K33‐, K48‐, and K63‐linked ubiquitin chains). After immunoprecipitation with anti‐HA magnetic beads, the ubiquitination of PKN2 was analyzed by immunoblotting. Cells were pretreated with 10 µ m MG132 for 6 h before harvesting (n = 3). (C) HEK‐293T cells were transfected with HA‐PKN2, Myc‐Ub, EV, Flag‐TRIM40, and Flag‐TRIM40‐29S, treated with 10 µ m MG132 for 6 h before harvesting. The ubiquitination level of PKN2 was detected by immunoblotting using an anti‐HA antibody (n = 3). (D) NRVMs were transfected with siRNA targeting TRIM40, followed by treatment with 1 µ m Ang II for 12 h. The phosphorylation level of PKN2 was detected by immunoblotting, with GAPDH used as the loading control (n = 3). (E) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel D (n = 3). (F) NRVMs were transfected with a TRIM40 expression vector, followed by treatment with 1 µ m Ang II for 12 h. The phosphorylation level of PKN2 was detected by immunoblotting, with GAPDH used as the loading control (n = 3). (G) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel F (n = 3). (H) NRVMs overexpressing Flag‐TRIM40 or Flag‐TRIM40‐C29S were treated with Ang II (1 µ m ) for 24 h. Phosphorylation of PKN2 at Ser815 (p‑PKN2) was detected by Western blot. GAPDH served as a loading control (n = 3). (I) Densitometric quantification of the Western blot bands from Figure (n = 3). (J) The level of p‐PKN2 was detected by immunoblotting in whole heart tissue lysates from mice infused with Ang II for 4 weeks (n = 6). (K) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel J (n = 6). (L) The level of p‐PKN2 was detected by immunoblotting in whole heart tissue lysates from mice subjected to TAC surgery (n = 6). (M) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel L (n = 6). (N) NRVMs overexpressing Flag‐TRIM40 or Flag‐TRIM40‐C29S were treated with Ang II (1 µ m ) for 24 h. Protein levels of MYH7, ANP and BNP were detected by Western blot. GAPDH served as a loading control (n = 3). (O) Densitometric quantification of the Western blot bands from Figure (n = 3). All quantitative data are presented as mean ± SEM. Data between two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among multiple groups were compared by one‐way ANOVA test, followed by Tukey post hoc test; ** p < 0.01.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Western Blot, Control, Immunoprecipitation, Magnetic Beads, Software, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: TRIM40 exacerbates Ang II‐induced cardiomyocyte hypertrophy and fibrosis by regulating PKN2. C57BL/6 mice received two injections of AAV9 encoding TRIM40 at one‐month intervals, followed by continuous infusion of saline or Ang II for two weeks. (A) Mouse systolic blood pressure was measured weekly using a non‐invasive tail‐cuff system (n = 6). (B) Serum Ang II concentration was detected in mice (n = 6). (C) Representative echocardiographic images of mice from each experimental group (n = 6). (D–F) Cardiac function parameters showing EF (D), FS (E), and IVRT (F) (n = 6). (G) Serum CK‐MB levels in mice (n = 6). (H) HW/BW of mice in each group (n = 6). (I) Representative freshly isolated heart specimens photographed against a white background (scale bar = 5 mm) (n = 6). (J) Cardiomyocyte size was assessed by fluorescein‐conjugated WGA staining (scale bar = 50 µm) (n = 6). (K) H&E staining of heart tissue sections (scale bar = 50 µm) (n = 6). (L, M) Myocardial fibrosis was evaluated by Masson's trichrome staining (L) and Picrosirius red staining (M) (scale bar = 50 µm) (n = 6). (N) Western blot analysis of MYH7, ANP, and BNP in myocardial tissues, with GAPDH as a loading control (n = 6). (O, P) mRNA expression levels of hypertrophy‐associated genes (O) and inflammation‐related genes (P) in myocardial tissues, normalized to Actb (n = 6). (Q) p‐PKN2 levels were detected by immunoblotting in whole heart lysates from mice infused with Ang II for 4 weeks (n = 6). (R) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel Q (n = 6). All quantitative data are presented as mean ± SEM. Data between the two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among multiple groups were compared by one‐way ANOVA test, followed by a Tukey post hoc test. ns indicates not statistically significant; * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Saline, Concentration Assay, Isolation, Staining, Western Blot, Control, Expressing, Software, Two Tailed Test

Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: TRIM40 exacerbates TAC‐induced cardiomyocyte hypertrophy and fibrosis by regulating PKN2. C57BL/6 mice received two injections of AAV9 encoding TRIM40 at one‐month intervals, followed by sham surgery or TAC surgery for two weeks. (A) Representative echocardiographic images of mice from each experimental group (n = 6). (B–D) Cardiac function parameters showing EF (B), FS (C), and IVRT (D) (n = 6). (E) Serum CK‐MB levels in mice (n = 6). (F) HW/BW of mice in each group (n = 6). (G) Representative freshly isolated heart specimens photographed against a white background (scale bar = 5 mm) (n = 6). (H) Cardiomyocyte size was assessed by fluorescein‐conjugated WGA staining (scale bar = 50 µm) (n = 6). (I) Cardiomyocyte hypertrophy evaluated by fluorescein‐conjugated WGA staining (scale bar = 50 µm) (n = 6). (J) H&E staining of heart tissue sections (scale bar = 50 µm) (n = 6). (K, L) Myocardial fibrosis was evaluated by Masson's trichrome staining (K) and Picrosirius red staining (L) (scale bar = 50 µm) (n = 6). (M) Western blot analysis of MYH7, ANP, and BNP in myocardial tissues, with GAPDH as a loading control (n = 6). (N, O) mRNA expression levels of hypertrophy‐associated genes (N) and inflammation‐related genes (O) in myocardial tissues, normalized to Actb (n = 6). (P) p‐PKN2 levels were detected by immunoblotting in whole heart lysates from mice infused with Ang II for 4 weeks (n = 6). (Q) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel P (n = 6). All quantitative data are presented as mean ± SEM. Data between the two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among multiple groups were compared by one‐way ANOVA test, followed by Tukey post hoc test; ** p < 0.01.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Isolation, Staining, Western Blot, Control, Expressing, Software, Two Tailed Test

Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: A Mechanism of TRIM40 Driving Cardiac Hypertrophy through PKN2 Ubiquitination.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Deducing the presence of proteins and proteoforms in quantitative proteomics

doi: 10.1038/s41467-018-04411-5

Figure Lengend Snippet: Select peptide-to-protein clusters with proteins differentially regulated in CFBE41o – vs. HBE41o – cells. a The protein cluster shows the family of related peroxiredoxin (PRDX) proteins. Edges and peptide node outlines in red as well as number associated with the edge in the peptide-to-protein clusters indicate differential expression according to the protein pair-centric analysis. b The protein cluster of serine–threonine kinase-related protein kinases PKN1, PKN2, and PKN3 is displayed. c The peptide-to-protein cluster of Na/K-ATPase proteins is depicted. It is expressed at the same levels in HBE41o – and CFBE41o – cells. d Western blot analysis was used to verify the difference in PKN2 expression levels between HBE41o – and CFBE41o – cells. Na + /K + -ATPase expression levels are shown as a loading control. Data in the western blot represent independent biological replicates, CFBE41o – : n = 2, HBE41o – : n = 2

Article Snippet: PKN2 and NaK-ATPase were detected by incubation with monoclonal anti-PKN2 antibody (dilution 1:1.000, clone 3A7, Novus Biologicals #H00005586-M01) and anti-Na + /K + ATPase antibody H-300 (dilution 1:2000, Santa Cruz #sc28800), respectively, followed by incubation with goat anti-mouse (dilution 1:10,000, Jackson ImmunoResearch Laboratories #205-035-108) or goat anti-rabbit (dilution 1:10,000, Cell Signaling Technology #7074S) IgG antibodies coupled to horse radish peroxidase, respectively.

Techniques: Quantitative Proteomics, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Protein Kinase PKN1 Represses Wnt/β-Catenin Signaling in Human Melanoma Cells *

doi: 10.1074/jbc.M113.500314

Figure Lengend Snippet: PKN1 and PKN2 siRNAs increase expression of AXIN2 in melanoma cell lines. The indicated cell lines were transiently transfected with siRNA oligonucleotides targeting PKN1 and PKN2 ( pan PKN ) or targeting nonsense sequence ( CTRL ). Forty-eight hours after transfection the cells were stimulated overnight with medium conditioned with WNT3A or with vehicle ( left panel , dose curve; right panel , single dose). The effects of WNT3A were assayed using quantitative PCR. cDNA was amplified using primers targeting GAPDH and the Wnt/β-catenin target gene AXIN2 . The number of copies of AXIN2 cDNA was averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. The error bars represent S.D., and the data in all plots are representative of at least two independent experiments.

Article Snippet: The following antibodies were used in this study: AKT (Cell Signaling Technologies, 2920), pAKT (Cell Signaling Technologies, 9271), LRP6 (Cell Signaling Technologies, 3395), pLRP6 (Cell Signaling Technologies, 2568), ERK (Cell Signaling Technologies, 4696), pERK (Cell Signaling Technologies, 9101), CTNNB1 (Cell Signaling Technologies, 2698), pCTNNB1 (Cell Signaling Technologies, 9561), PARP1 (Cell Signaling Technologies, 9546), PKN1 (Santa Cruz Biotechnology, sc-136037), PKN2 (Bethyl Laboratories, A302-443A), pPKN (Cell Signaling Technologies, 2611), HA (Roche Applied Science, 3F10), GFP (Abcam, ab290), HSP90 (Abcam, ab13494), β-tubulin (Sigma-Aldrich, T7816), and AnnexinV (eBioscience, 88-8007-72).

Techniques: Expressing, Transfection, Sequencing, Real-time Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Yersinia Virulence Factor YopM Induces Sustained RSK Activation by Interfering with Dephosphorylation

doi: 10.1371/journal.pone.0013165

Figure Lengend Snippet: Peptides identified by peptide mass fingerprint analysis.

Article Snippet: Clones for in vitro translation have been obtained from ImaGenes GmbH (Berlin, Germany), except PKN1 for which the above mentioned expression clone was used: RSK1 (gi:15929012), RSK2 (gi:109730041), RSK3 (gi:27696716), RSK4 (gi:32450548), PKN2 (gi:30354737), PKN3 (gi:38565959).

Techniques: