Journal: eLife

Article Title: In vivo proximity labeling identifies cardiomyocyte protein networks during zebrafish heart regeneration

doi: 10.7554/eLife.66079

Figure Lengend Snippet: ( A ) ErbB2-BirA2-HA-P2A-GFP is expressed in cardiomyocytes via the cmlc2 promoter. ( B ) Summary of experiment and timeline. cmlc2:erbb2-birA2-HA-P2A-GFP ventricles were collected as uninjured samples or 14 days after induced cardiomyocyte ablation (dpi). ( C ) Known direct interactors of ErbB2 that were captured in the ErbB2 BioID2 assay. 108 proteins showed a change >1.5-fold when normalized to uninjured cmlc2:erbb2-birA2-HA-P2A-GFP ventricles. These data were analyzed for known ErbB2 interactors. Colors and size of interactor correspond to fold changes identified during regeneration. Green: no change; blue: levels decrease; yellow: levels increase. Tns1: tensin 1 (1.57-fold); Hspb2: heat shock protein beta 2 (−2.43-fold); Gapdh: glyceraldehyde 3-phosphate dehydrogenase (p>0.05); Acta1b: actin alpha 1 (p>0.05); Ezrb: ezrin b (p>0.05); Ctnnd1: catenin δ1 (−4.59-fold); RhoA-b: Rho A-b (13-fold); Scrib: scribble (p>0.05); Anxa2a: annexin A2a (7.31-fold). ( D ) Over-representation test – pathway analysis of proteins increased 1.5-fold or more in ErbB2-BirA data set. p<0.001, FDR < 0.005%. Fold enrichment is shown, and protein number in red. ( E ) Co-immunoprecipitation of ErbB2-BirA2-HA from uninjured or regenerating cmlc2:erbb2-birA2-HA-P2A-GFP hearts. Rho A association with ErbB2 is increased after injury. Anti-HA antibody was used for ErbB2 detection, and troponin T and IgG were used as loading controls. ( F ) Analysis of known Rho A interactors in BirA2-GFP BioID2 data set. All known direct interactors were found to be increased when normalized to uninjured hearts. Size of circles indicates fold change; proteins are sorted clockwise after fold change from high to low. Scrib: scribble (90.9-fold); ErbB2: Erb-b2 receptor tyrosine kinase 2 (52.9-fold); Gna13: guanine nucleotide binding protein alpha 13a (25.8-fold); Rtn4a: reticulon 4a (8.82-fold); ROCK2a: Rho-associated, coiled-coil containing protein kinase 2a (5.05-fold); Ilk: integrin-linked kinase (3.91-fold); Pfn1: profilin 1 (3.25-fold); Arhgap21b: Rho GTPase activating protein 21b (2.65-fold); Wasla: WASP-like actin nucleation promoting factor a (2.5-fold); Lrp5: low-density lipoprotein receptor-related protein 5 (2.21-fold); Ctnnd1: catenin δ1 (2.2-fold); Msna: moesin a (1.9-fold). ( G ) Western blot analysis of ROCK2 levels in uninjured and regenerating hearts. ROCK2 levels are increased during heart regeneration.

Article Snippet: Antibody , Anti-ROCK2 (rabbit polyclonal) , Bioss Antibodies , Cat#: BS-1205R , IF: 1:500.

Techniques: Immunoprecipitation, Binding Assay, Western Blot

Journal: eLife

Article Title: In vivo proximity labeling identifies cardiomyocyte protein networks during zebrafish heart regeneration

doi: 10.7554/eLife.66079

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-ROCK2 (rabbit polyclonal) , Bioss Antibodies , Cat#: BS-1205R , IF: 1:500.

Techniques: Transgenic Assay, Recombinant, Plasmid Preparation

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet: FAK translocation, ROCK remodulation, and activation of proliferation pathway (A) Estimation of uptake of α-SYN-TMR protofibrils by U251 cells treated with 1μM α-SYN protofibrils and pre-treated (before 30min) with 0.5 μM cytochalasin D and 5 μM Y-27632 (ROCK inhibitor) by flow cytometry. (B) Quantification of percentage gated fluorescence intensity of α-SYN-TMR protofibrils in the above experiment. (C) Fluorescence images of maximum intensity projected confocal z-stacks in astrocytes stained with Phalloidin (green) and FAK (red). The upper panel has nucleus stained with DAPI and the lower panel is without DAPI. White arrows indicate the nuclear colocalization of FAK, red arrows indicate TNTs and pink arrows indicate FAK at focal adhesion. (D) Quantification of FAK intensity in the nucleus of the cells with and without TNTs. (E and G) Fluorescence images of maximum intensity projected confocal z-stacks in U-87 MG cells stained with FAK (red) and pFAK (red) with DAPI, respectively. (F and H) Quantification of FAK and pFAK fluorescence intensity per cell in the nucleus. Quantifications are done from 15 image frames in a set, and each image frame has 15–20 cells. (I) Western blot images showing the change in ROCK1 and ROCK2 expressions with time on 1μM α-SYN protofibril treatment in U-87 MG cells. (J) Quantification of ROCK1 and ROCK2 western blots. Full-length western blots of four repeats are represented in Figure S9 . (K) Western blot images showing increased intensity of pERK1/pERK2, NF-κB, and Cdk1 with time on 1μM α-SYN protofibril treatment in U-87 MG cells. Full-length western blots of three repeats for pERK1/pERK2 and NF-κB and five repeats for Cdk1 were represented in Figure S10 . (L) Quantification of pERK1/pERK2, NF-κB, and Cdk1 western blots. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗ p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. n = 3.

Article Snippet: Later the suspension was spun at 12,000 rpm for 10 min and the supernatant was collected and stored at -20 0 C. After normalizing the protein concentration, western blot assay was performed with primary antibodies GAPDH Mouse (Cloud-Clone # CAB932Hu22 dilution 1:1000), ROCK1 (CST # 4035T dilution 1:1000), ROCK2 (CST # 8236S dilution 1:1000), NF-kB p65/RelA Mouse mAb (Abclonal # A10609 dilution 1:1000), phospho ERK1-T202 + ERK2- T18 (Abclonal # AP0485 dilution 1:1000), Cdk1 Rabbit pAb (Abclonal #A0220 dilution 1:1000), p21 Waf1/Cip1 (12D1) Rabbit mAb (CST #2947 dilution 1:1000) and secondary antibodies, Goat anti-mouse (H+L) (Invitrogen #32430 dilution 1:1500) and Goat Anti-Rabbit (H+L) (Invitrogen #32460 1:1500).

Techniques: Translocation Assay, Activation Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet:

Article Snippet: Later the suspension was spun at 12,000 rpm for 10 min and the supernatant was collected and stored at -20 0 C. After normalizing the protein concentration, western blot assay was performed with primary antibodies GAPDH Mouse (Cloud-Clone # CAB932Hu22 dilution 1:1000), ROCK1 (CST # 4035T dilution 1:1000), ROCK2 (CST # 8236S dilution 1:1000), NF-kB p65/RelA Mouse mAb (Abclonal # A10609 dilution 1:1000), phospho ERK1-T202 + ERK2- T18 (Abclonal # AP0485 dilution 1:1000), Cdk1 Rabbit pAb (Abclonal #A0220 dilution 1:1000), p21 Waf1/Cip1 (12D1) Rabbit mAb (CST #2947 dilution 1:1000) and secondary antibodies, Goat anti-mouse (H+L) (Invitrogen #32430 dilution 1:1500) and Goat Anti-Rabbit (H+L) (Invitrogen #32460 1:1500).

Techniques: Recombinant, Staining, Software

Journal: iScience

Article Title: Targeting SRSF2 mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure

doi: 10.1016/j.isci.2024.109443

Figure Lengend Snippet:

Article Snippet: Membranes were blocked in 5% (w/vol) milk powder in 0.05% Tween-20/TBS (TBS-T) and probed for 16 hours at 4°C with the indicated antibodies: rabbit anti-ROCK1 (C8F7) (Cell Signaling Technology, CST-4035S), rabbit anti-Rock2 (E5T5P) (Cell Signaling Technology, CST-47012S), and rabbit anti-Gapdh (D16H11) (Cell Signaling Technology, #5174).

Techniques: Selection, Sequencing

Journal: Oncotarget

Article Title: Phosphorylation of Nucleophosmin at Threonine 234/237 is associated with HCC metastasis

doi:

Figure Lengend Snippet: A. Immunoprecipitation assays revealed a physical interaction between NPM and ROCK2. B. The protein expression of ROCK2 in NPM-wild type (NPM WT) and NPM Thr234/237A mutant was comparable to control. C. Knockdown of CDK1 did not change the protein expression of ROCK2 when compared to NTC. D. NPM Thr234/237A mutate reduced phosphorylation of LIM kinase (LIMK), a substrate of ROCK2, in Hep3B and Huh7 transfected cells. E. Similar results were observed when CDK1 was knocked down in Bel 7402 and SMMC cells. F. NPM WT transfectants exhibited increased stress fiber network. Stress fibers (polymerized actin) and actin filaments were demonstrated by phalloidin (red), while NPM Thr234/237A mutant exhibited loss of stress fibers, when compared with NPM WT. (magnification × 63).

Article Snippet: Specimens were incubated with NPM (B0556, Sigma), p-NPM-Thr 234/237 (619102, Biolegend) and ROCK2 (21645-1-AP, Proteintech) in a dilution of 1:100.

Techniques: Immunoprecipitation, Expressing, Mutagenesis, Control, Knockdown, Phospho-proteomics, Transfection

Journal: Open Biology

Article Title: SLMAP3 is crucial for organogenesis through mechanisms involving primary cilia formation

doi: 10.1098/rsob.240206

Figure Lengend Snippet: Primary antibodies used for western blot analysis.

Article Snippet: Phospho-ROCK2 (S1366) , GeneTex , GTX122651 , rabbit , 1:750.

Techniques: Western Blot