Journal: Cell Discovery

Article Title: A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A

doi: 10.1038/celldisc.2016.14

Figure Lengend Snippet: Acetylation of lysine 150 on Cdc25A negatively regulates the G2/M checkpoint in response to IR. ( a ) Knockout of endogenous Cdc25A in HeLa cells through CRISPR-Cas9. ( b ) Re-expression of wild-type (WT) or K150R mutant Cdc25A in the Cdc25A knockout cell line through pSIN lentivirus. ( c, d ) Degradation rate of WT-Cdc25A and K150R-Cdc25A mutant post IR (6 Gy) using western blot. ( e, f ) Cdc25A KO HeLa cells stably re-expressing WT-Cdc25A or K150R-Cdc25A mutant were exposed to IR (6 Gy). The cells were harvested and analyzed using flow cytometry after incubation with 100 ng ml −1 of nocodazole for 10 h. The percentage of cells positive for phospho-histone H3 (p-H3) is indicated. N =3. Bars indicate the s.e.m. *** P <0.001, Student’s t- test.

Article Snippet: After the cells were centrifuged, the cell pellet was suspended in 100 ml of PBS containing 1% bovine serum albumin and 0.75 μg of a polyclonal antibody that specifically recognizes the phosphorylated form of histone H3 (Upstate Biotechnology, Lake Placid, NY, USA), and incubated for 1.5 h at room temperature.

Techniques: Knock-Out, CRISPR, Expressing, Mutagenesis, Western Blot, Stable Transfection, Flow Cytometry, Incubation

Journal: BMC Cell Biology

Article Title: Evidence for the involvement of G i2 in activation of extracellular signal-regulated kinases in hepatocytes

doi: 10.1186/1471-2121-2-13

Figure Lengend Snippet: Western analysis of the effects of ribozyme on G i2α and ERK1/2. G i2α ribozyme (Rz) or non-cleaving G i2α ribozyme (Rzm) complexed with DOTAP giving final ribozyme concentrations of 2.5 μM, or only DOTAP (Ctr) were added to hepatocyte cultures at 4–5 hours after the time of seeding. A: Expression of G i2α protein was assessed after 45 h of ribozyme treatment using antibody (from Calbiochem) directed against C-terminal end of G i1/i2α . B: Expression of G i2α and G qα protein levels in the same samples subsequent to 30 h of ribozyme treatment using antibodies (from NEN™ Life Science Products) against C-terminal sequences of G i1/i2α or G qα , respectively. The polyclonal antibodies used to assess G i2α recognize both the α subunit of G i2 and G i1 . As shown previously hepatocytes do not express G i1α , so the reactivity with these antibodies reflects only the G i2α levels. C, D: After 45 h of ribozyme treatment cells were stimulated with or without PGF 2α (10 μM) for 5 min before they were harvested. Immunoblot using antibody against dually phosphorylated ERK1/2 (i.e. ERK1/2-P) (C) is depicted. In Fig. D is developed images from the same immunoblot using antibody detecting total amount of ERK1/2 (i.e. both phosphorylated and unphosphorylated forms) (upper panel) and antibody against dually phosphorylated fractions of ERK1/2 (lower panel).

Article Snippet: Aliquots with 20 μg cell protein (total cell lysate prepared in Laemmli buffer) were electrophoresed on 10 % polyacrylamide gels (acrylamide:N'N'-bis-methylene acrylamide 30:0.8) followed by protein electrotransfer to nitrocellulose membranes and immunoblotting with a polyclonal MAP kinase antibody against the dually threonine- and tyrosine phosphorylated forms of ERK1 (p44 mapk ) and ERK2 (p42 mapk ) or an antibody detecting both the phosphorylated and unphosphorylated forms (Promega Corporation, Madison, WI, USA).

Techniques: Western Blot, Expressing

Journal: Journal of Cell Science

Article Title: ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters

doi: 10.1242/jcs.215517

Figure Lengend Snippet: Mutation of ADAP Y595 abolishes SLP-76 microcluster movement and persistence. (A) Schematic depiction of ADAP-120 chimeras with tyrosine to phenylalanine mutations at positions 595, 625 and 651. (B) ADAP-deficient (JDAP) cells transiently expressing the indicated 3×Flag.TRT-tagged chimeras and SLP-76.WT.mCFP were stimulated, imaged and presented as in Fig. 2 (n=3 experiments for all conditions except WT, which was n=4 experiments; see Table S3 for cell numbers). Scale bars: 10 μm (main images); 5 μm (horizontal); 60s (vertical) (kymographs). (C) For the conditions in B, SLP-76 microcluster trajectories were manually traced in iVision and average trajectories are shown as composite kymographs. The x- and y-axes depict centripetal displacement over time and intensity encodes the fraction of clusters persisting; arrowheads indicate the time at which half of the microclusters had dissociated. Microcluster properties, number of cells analyzed, and statistical comparisons are listed in Table S3.

Article Snippet: Rabbit antibodies targeting the Y595 phosphorylated and Y595 non-phosphorylated forms of ADAP were developed at Pacific Immunology (Ramona, CA) using the synthetic immunogen EDDQEVpYDDVAEQD.

Techniques: Mutagenesis, Expressing

Journal: Journal of Cell Science

Article Title: ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters

doi: 10.1242/jcs.215517

Figure Lengend Snippet: Multivalent interactions of ADAP with SLP-76 stabilize ADAP phosphorylation. (A) J14.SY cells were stably transduced with lentiviruses encoding the indicated 3×Flag.TRT.ADAP-120 chimeras. Parental and transduced cells were either left unstimulated or stimulated with C305 for 2 min. Total lysates were western blotted (WB) as indicated. FLAG serves as a loading control while pY145 SLP-76 indicates TCR activation. (B) J14.SY cells stably expressing the indicated ADAP chimeras were left unstimulated or stimulated with pervanadate and C305 for 10 min. Total lysates were western blotted for FLAG, total ADAP or ADAP pY595. (C) J14.SY cells stably expressing 3×FLAG.TRT.ADAP were stimulated with C305 and lysed. Y595-phosphorylated ADAP (pY595) and total ADAP (FLAG) were immunoprecipitated (IP) using fixed amounts of each antibody and oversaturating amounts of lysate. This ensured that the total amount of ADAP captured by the pY595 antibody was fixed, regardless of the time of stimulation. Each FLAG IP contained 30 times more total ADAP than the corresponding pY595 IP. Equal amounts of total ADAP were loaded and western blotted for ADAP, SKAP55, and total phosphotyrosine (pY). ex., exogenous; en., endogenous. (D) Parental J14 cells or J14 cells stably reconstituted with WT (WT) or SH2 mutant (RK) SLP-76.YFP chimeras were stimulated, lysed and western blotted for ADAP pY595. pY783 PLCγ1 serves as a control for TCR signaling. All western blots are representative of three or more independent experiments. (A–D) For all western blots, the SLP-76.YFP chimera migrates just above the 100 kDa marker, endogenous ADAP migrates just above the SLP-76 chimera and 3xFlag-TRT-tagged ADAP chimeras migrate just below 150 kDa; endogenous SKAP55 migrates just below 50 kDa. Time of stimulation is given in minutes. (E) Model for the SLP-76 SH2 domain-dependent maintenance of ADAP phosphorylation: TCR ligation results in the Lck-dependent activation of ZAP-70, triggering the formation of oligomers containing LAT, Gads and SLP-76 (top right). Concurrently, small populations of ADAP become phosphorylated by a Src kinase (green arrow). Heavily phosphorylated ADAP species capture LAT–SLP-76 oligomers via multivalent interactions between the SLP-76 SH2 domain and tyrosine residues 595, 651 and 771 of ADAP (red circles), shielding ADAP from dephosphorylation (red blocking arrow). In contrast, partially phosphorylated ADAP is de-phosphorylated by cytoplasmic phosphatases (blue arrow). The sites of other tyrosine-phosphorylated residues in ADAP are based on mass spectroscopy studies (pale circles) (Lange et al., 2010; Sylvester et al., 2010).

Article Snippet: Rabbit antibodies targeting the Y595 phosphorylated and Y595 non-phosphorylated forms of ADAP were developed at Pacific Immunology (Ramona, CA) using the synthetic immunogen EDDQEVpYDDVAEQD.

Techniques: Phospho-proteomics, Stable Transfection, Transduction, Western Blot, Control, Activation Assay, Expressing, Immunoprecipitation, Mutagenesis, Marker, Ligation, De-Phosphorylation Assay, Blocking Assay, Mass Spectrometry

Journal: Journal of Cell Science

Article Title: ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters

doi: 10.1242/jcs.215517

Figure Lengend Snippet: Tyrosine phosphorylation controls the localization of ADAP between the actin-rich leading edge and SLP-76 microclusters. (A) J14.SY cells stably expressing 3×Flag.TRT.ADAP-120 were co-stimulated with C305 and pervanadate for the indicated times. Total lysates were western blotted (WB) with custom antisera specific for phosphorylated or non-phosphorylated Y595 (pY595 and non-pY595, respectively) or with an anti-FLAG control antibody. The dominant bands are exogenous (upper) and endogenous (lower) ADAP. The image shown is representative of three independent experiments. (B) J14.SY cells stably expressing exogenous 3×Flag.TRT.ADAP-120 were stimulated as in Fig. 1 and fixed after 10 min. Fixed cells were stained with ADAP antisera targeting phosphorylated pY595 (n=3 experiments, 33 cells) or non-pY595 (n=3 experiments, 42 cells). (C) J14.SY cells without exogenous ADAP expression were stimulated as in Fig. 1 and fixed after 10 min. Fixed cells were stained with ADAP antisera targeting pY595 (n=3 experiments, 65 cells) or non-pY595 (n=3 experiments, 34 cells). (D) J14.SY cells stably expressing 3×Flag.TRT.ADAP-120 were stimulated as in Fig. 2 and fixed. Before imaging, cell bodies were left intact (upper row) or sheared away, leaving only the cell footprint (middle row). In the bottom row, J14.SY cells without exogenous ADAP expression were sheared away from the substrate prior to immunofluorescence staining of the residual adherent structures with pY595 sera (n=2 experiments; 31 WT unsheared cells, 54 WT sheared cells, 19 J14.SY sheared cells). Scale bars: 10 µm.

Article Snippet: Rabbit antibodies targeting the Y595 phosphorylated and Y595 non-phosphorylated forms of ADAP were developed at Pacific Immunology (Ramona, CA) using the synthetic immunogen EDDQEVpYDDVAEQD.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Western Blot, Control, Staining, Imaging, Immunofluorescence

Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on cardiac muscle fibrosis and hypertrophy. ( A ) Picro-Sirius Red staining. Scale bar = 200 µm. ( B ) Quantification of Picro-Sirius Red staining. mRNA levels of ( C ) αSMA and ( D ) TGF-β1, two markers of fibrosis. ELISA assays were used to quantify ( E ) TGF-β and ( F ) the active phosphorylated form of SMAD2 (P¬SMAD2), a transcription factor mainly involved in TGF-β signalling. mRNA levels of ( G ) BNP and ( H ) ANP, two markers of hypertrophy. ( I ) ELISA assay was also used to quantify the levels of ANP. The percentage of stained areas was calculated in cardiac muscle sections, and the subsequent ratios are presented as relative expressions to WT values. mRNA levels were normalised to cyclophilin, and the subsequent ratios were presented as relative expressions to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on ApN receptors and signalling in the dystrophic cardiac muscle. mRNA levels of ( A ) AdipoR1 and ( B ) AdipoR2, adiponectin main receptors. ELISA assays were used to quantify ( C ) AdipoR1 and ( D ) AdipoR2. ( E ) The ratio of AdpoR1 over AdipoR2 mRNA levels was calculated within the cardiac muscle. ELISA assays were used to quantify ( F ) the active phosphorylated form of AMPKα (P-AMPK), ( G ) calcium/calmodulin-dependent protein kinase 2 (CAMKK2), and ( H ) peroxisome proliferator-activated receptor alpha (PPARα), ApN/AdipoRon, main signalling pathways in muscle. ( I ) mRNA levels of PGC-1α. ELISA assays were used to quantify ( J ) PGC-1α, ( K ) the active phosphorylated form of the p65 subunit of NF-κB (P-p65), a transcription factor mainly involved in inflammation, and ( L ) utrophin A (UTRN), a dystrophin analogue. mRNA levels were normalised to cyclophilin, and the subsequent ratios are presented as relative expression to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. WT mice. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on cardiac muscle oxidative capacity, injury, and overall muscle function. mRNA levels of ( A ) ERRα and ( B ) mtTFA, two markers of mitochondrial biogenesis. ELISA assay was used to quantify ( C ) TOMM20, a marker of mitochondrial content. ( D ) Wire test where mice hanging time was recorded (s). ( E ) Fore-limb grip test and ( F ) fore- and hind-limb grip test, measuring muscle strength expressed in Gram-force relative to body weight (gf/gBW). ( G ) Treadmill running exercise, where the total distance covered on the third day was measured (m). ( H ) CK and ( I ) LDH plasma activities assessing muscle injury and expressed as IU/L. ( J ) ELISA assay was used to quantify the active phosphorylated form of RIP (P-RIP), an important regulator of cellular stress that triggers a regulated pathway for necrotic cell death called necroptosis. mRNA levels were normalised to cyclophilin, and the subsequent ratios are presented as relative expressions to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all ex vivo experiments. Data are means ± SD; n = 7–8 mice per group for all in vivo functional tests. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Marker, Ex Vivo, In Vivo, Functional Assay