dispase  (Worthington Biochemical)

Journal: The FASEB Journal

Article Title: Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R

doi: 10.1096/fj.201802338RR

Figure Lengend Snippet: Overexpression of Tsg101 enhances recycling of IGF-1R in cardiomyocytes. A , B ) Representative immunoblots ( A ) and quantitative analyses ( B ) of streptavidin immunoprecipitates (IP), visualized using an IGF-1R antibody. Whole-cell lysates (WCLs) were used as loading controls. Four independent experiments were performed for recycling assays. C ) Representative images of immunofluorescence staining with streptavidin Alexa Fluor 594 conjugate after IGF-1R biotin was internalized and recycled in Ad.Tsg101 and Ad.GFP NRCMs; n = 4 plates, 25–30 cells per plate. Scale bars, 10 μm. * P

Article Snippet: NRCMs were isolated using the Worthington Neonatal Cardiomyocytes Isolation System (Worthington Biochemical) according to the manufacturer’s procedures.

Techniques: Over Expression, Western Blot, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R

doi: 10.1096/fj.201802338RR

Figure Lengend Snippet: Overexpression of Tsg101 in NRCMs promotes cell hypertrophy. A ) Diagrams depicting the recombinant adenoviral vectors Ad.GFP and Ad.Tsg101. B ) Representative images showing Ad.GFP- or Ad.Tsg101-infected NRCMs (left panel is under bright field; right panel is the same field under fluorescence microscope). Scale bars, 20 μm. C ) Western blot showing increased expression of Tsg101 in Ad.Tsg101 cardiomyocytes compared with Ad.GFP cells. D , E ) Measurement of myocyte cross-sectional area in those infected cells (green) that were stained with cardiomyocyte-specific α-actinin antibody (red). Scale bars, 20 μm. n = 6 plates, 25–30 cells per plate. F , G ) Representative immunoblots ( F ) and quantitative analyses ( G ) showing expression of PM IGF-1R, total IGF-1R, Akt phosphorylation at Ser473, Rab5a, Rab4a, Rab7, Rab11a, and FIP3 in Ad.Tsg101 and Ad.GFP cells. GAPDH was used as loading control for total protein, total Akt was loading control for Akt phosphorylation, and Na/K-ATPase was loading control for PM protein. PM, plasma membrane; n = 4 for independent experiments. * P

Article Snippet: NRCMs were isolated using the Worthington Neonatal Cardiomyocytes Isolation System (Worthington Biochemical) according to the manufacturer’s procedures.

Techniques: Over Expression, Recombinant, Infection, Fluorescence, Microscopy, Western Blot, Expressing, Staining

Journal: The FASEB Journal

Article Title: Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R

doi: 10.1096/fj.201802338RR

Figure Lengend Snippet: Tsg101-mediated cardiomyocyte hypertrophy is attenuated by monensin and KD of FIP3. A , B ) Treatment of NRCMs with monensin (1 μM) 1 h prior to adenovirus infection resulted in reduced plasma membrane levels of IGF-1R; n = 4 for independent experiments; * P

Article Snippet: NRCMs were isolated using the Worthington Neonatal Cardiomyocytes Isolation System (Worthington Biochemical) according to the manufacturer’s procedures.

Techniques: Infection

Journal: The FASEB Journal

Article Title: Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R

doi: 10.1096/fj.201802338RR

Figure Lengend Snippet: Diagram showing structural components and major players involved in the Tsg101-mediated endosomal recycling of IGF-1R. Tsg101 interacts with and enhances the function of FIP3 at the ERC. Increased activity of the ERC promotes translocation of IGF-1R to the plasma membrane, which in turn augments IGF-IR and Akt signaling in cardiac myocytes, leading to physiologic cardiac hypertrophy.

Article Snippet: NRCMs were isolated using the Worthington Neonatal Cardiomyocytes Isolation System (Worthington Biochemical) according to the manufacturer’s procedures.

Techniques: Activity Assay, Translocation Assay

Journal: bioRxiv

Article Title: Multiomics reveals persistence of obesity-associated immune cell phenotypes in adipose tissue during weight loss and subsequent weight regain

doi: 10.1101/2021.08.20.455954

Figure Lengend Snippet: Adipose tissue immune remodeling during WL and WC. Body weight gain results in a transition from abundant type 2 immune cells present in lean adipose tissue towards an accumulation of immune cells/phenotypes associated with lipid handling (lipid associated macrophages, LAMs) and antigen presentation. WL does not restore the antigen presentation phenotype and is associated with T cell exhaustion. T cell exhaustion and LAMs are further amplified by weight regain. There is a di sconnect between systemic glucose homeostasis and immune remodeling in adipose tissue, such that WL corrects obesity-induced glucose intolerance, but does not resolve the altered immune cell composition caused by diet-induced obesity.

Article Snippet: Stromal Vascular Fraction Isolation and Cell Sorting Mice were euthanized by isoflurane overdose and cervical dislocation and perfused with 20 ml PBS through the left ventricle. eAT pads were collected, minced, and digested in 6 ml of 2-mg/mL type II collagenase (Worthington # LS004177) for 30 min at 37°C.

Techniques: Amplification

Journal: Nature Communications

Article Title: Targeting VEGF-A in myeloid cells enhances natural killer cell responses to chemotherapy and ameliorates cachexia

doi: 10.1038/ncomms12528

Figure Lengend Snippet: Chemerin release and NK cell anti-tumour defense account for the improved growth restriction on cisplatin treatment in Mut (LysMCre/VEGF f / f ) mice. ( a ) Percentage of NK1.1-positive cells in cisplatin-treated tumours from WT and Mut mice that received i.p. injections of PBS as control (upper panel) or chemerin-neutralizing antibody (lower panel), determined by flow cytometry. Tumours were analysed in three independent experiments. ( b ) Representative micrographs of SA-β-gal-activity at pH 6.0 on day 18 in tumour sections after treatment with CDDP alone (upper panel), with CDDP and chemerin-neutralizing antibody (middle panel) or with CDDP and NK-depleting antibody mAB PK136 (lower panel). ( c ) Quantification of SA-β-gal-positive cells in b (untreated, n ≥4; CDDP, n ≥6). ( d ) Tumour volumes on day 18 after treatment with CDDP alone, CDDP and chemerin-neutralizing antibody, CDDP and mAB PK136 or CDDP and intratumoural injections of recombinant chemerin ( n ≥5). Bars represent mean values; error bars indicate the s.e.m.; statistical significance was determined by one-way analysis of variance followed by Bonferroni post-hoc test when more than two groups were compared. Statistical significance is indicated as * P

Article Snippet: Flow cytometry analysis Tumours were digested with 0.1% collagenase type III (Worthington) and 1 U ml−1 DNAse I (Promega) and incubated at 37 °C for 1 h. One hundred and six tumour cells were incubated with Fc-Block (BD Biosciences, #553141 at 1/100) before labelling with fluorochrome-conjugated antibodies.

Techniques: Mouse Assay, Flow Cytometry, Cytometry, Activity Assay, Recombinant

Journal: Nature methods

Article Title: The proteomes of transcription factories containing RNA polymerases I, II or III

doi: 10.1038/nmeth.1705

Figure Lengend Snippet: Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c) “Run-on” activity assayed later during fractionation (as in a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by

Article Snippet: Resuspended nuclei were digested (30 min; 33°C) with either (i) DNase I (protease- and RNase-free; Worthington;10 units/107 cells in 100 μl PB plus 0.5 mM CaCl2 ), or (ii) Hae III (Invitrogen; 1000 units/107 cells), or (iii) Hind III (New England Biolabs; 1000 units/107 cells) in PB; reactions were stopped by adding EDTA to 2.5 mM and cooling in iced water.

Techniques: Purification, Mass Spectrometry, Activity Assay, Fractionation