Journal: Journal of Neuroscience

Article Title: Inactivation of Socs3 in the Hypothalamus Enhances the Hindbrain Response to Endogenous Satiety Signals via Oxytocin Signaling

doi: 10.1523/jneurosci.1669-12.2012

Figure Lengend Snippet: Figure 1. Efficiency of Cre-mediated recombination and validation of functional Socs3 inactivation. A, Representative pictures showing typical -gal immunohistochemical staining (red) obtained 2 weeks after Ad-Cre delivery in the MBH of 3-month-old, ROSA26R male mice. Cre-mediated recombination is extensively detected in Hu C/D-positive neurons (green) in the ARC, VMH, andDMH(top).However,virtuallyno-galstainingisdetectedinthePVN(bottom).3V,3rdventricle;AHP,anteriorhypothalamicarea,posteriorpart;DM,dorsomedialhypothalamicnucleus;ME, medianeminence;VMHDM,dorsomedialpartoftheVMH;VMHVL,ventrolateralpartoftheVMH.B,NeuronstransducedwithAd-CreintheMBHshowleptin-inducedStat3Y705-phosphorylation (pStat3,brown)inmiceinjectedwithleptin(1mg/kg;i.p.)andperfused1hlater.TransducedneuronswerealsostainedwithX-gal(blue)toreveal-galactivity.C,D,WesternblotanalysesofSocs3 and-actininMBHlysatesofWTandSocs3 MBHmice.Micewerefastedovernightandtissueswereprocessed5haftervehicle(Veh)orleptininjection(3.5mg/kg;i.p.).EachlaneinCrepresents one animal. In D, Socs3 expression normalized to -actin levels, quantified by densitometry (n 6–8 animal per group). Data are mean SEM **p 0.004, Mann–Whitney test. NS, nonsignificant. Scale bars: 100 m.

Article Snippet: Membranes were then blocked in 20 mM Tris buffer, pH 7.4, containing 5% bovine serum albumin and 0.1% Tween 20 and incubated with a rabbit polyclonal anti-Socs3 antibody (1:1000; Cell Signaling Technology) overnight at 4°C.

Techniques: Biomarker Discovery, Functional Assay, Immunohistochemical staining, Staining, Phospho-proteomics, Expressing, MANN-WHITNEY

Journal: Journal of Neuroscience

Article Title: Inactivation of Socs3 in the Hypothalamus Enhances the Hindbrain Response to Endogenous Satiety Signals via Oxytocin Signaling

doi: 10.1523/jneurosci.1669-12.2012

Figure Lengend Snippet: Figure 3. MBH Socs3-deficient mice display an increased response to exogenous leptin. Leptin sensitivity was determined by measuring 24 h food intake on the second day of treat- ment with exogenous leptin (1 mg/kg; i.p.), administered twice daily (n 8 WT and n 10 Socs3 MBH mice). Data are mean SEM. *p 0.05 and ***p 0.001, determined by Bonferronimultiple-comparisontestfollowingrepeated-measures,two-wayANOVA.NS,non- significant; Veh, vehicle; Lep, leptin.

Article Snippet: Membranes were then blocked in 20 mM Tris buffer, pH 7.4, containing 5% bovine serum albumin and 0.1% Tween 20 and incubated with a rabbit polyclonal anti-Socs3 antibody (1:1000; Cell Signaling Technology) overnight at 4°C.

Techniques:

Journal: Journal of Neuroscience

Article Title: Inactivation of Socs3 in the Hypothalamus Enhances the Hindbrain Response to Endogenous Satiety Signals via Oxytocin Signaling

doi: 10.1523/jneurosci.1669-12.2012

Figure Lengend Snippet: Figure 4. MBH Socs3 deficiency triggers an increased hindbrain response to endogenous meal-relatedsatietysignals.A,Cumulativeshort-termfoodintakeatthebeginningofthedark phase measured in nonfasted WT (n 11) and Socs3 MBH mice (n 10) at 30 min, 1 h, and 2hafterlightsoff.B,Nocturnal,2hfoodintakeinnonfastedWT(n12)andSocs3 MBHmice (n9)measuredafterinjectionofvehicle(Veh)ordevazepide(1mg/kg;i.p.),anantagonistof CCK-Areceptors.C,Afteranovernightfast,30minfoodintakewasmeasuredinWT(n8)and Socs3 MBH mice (n 6) injected with leptin (Lep; 0.5 mg/kg; i.p.) or vehicle (Veh), in combi- nationwithCCK(2g/kg;i.p.)orvehicle.DataaremeanSEM.Two-way,repeated-measures ANOVA, followed by Bonferroni multiple-comparison test was used in A–C, where *p 0.05 and **p 0.01. In C, ##p 0.01 versus vehicle-treated Socs3 MBH mice, determined by Bonferroni multiple-comparison test following two-way, repeated-measures ANOVA. NS, nonsignificant.

Article Snippet: Membranes were then blocked in 20 mM Tris buffer, pH 7.4, containing 5% bovine serum albumin and 0.1% Tween 20 and incubated with a rabbit polyclonal anti-Socs3 antibody (1:1000; Cell Signaling Technology) overnight at 4°C.

Techniques: Injection, Comparison

Journal: Journal of Neuroscience

Article Title: Inactivation of Socs3 in the Hypothalamus Enhances the Hindbrain Response to Endogenous Satiety Signals via Oxytocin Signaling

doi: 10.1523/jneurosci.1669-12.2012

Figure Lengend Snippet: Figure 5. Hindbrain oxytocin signaling mediates the increased sensitivity to satiety signals in Socs3 MBH mice. A, Nocturnal food intake in nonfasted WT (n 6) and Socs3 MBH mice (n 7) measured 2 h after 4V injection of vehicle (Veh) or SSR126768A (SSR), an oxytocin receptor antagonist. B, Mature oxytocin tissue content in the DVC of WT and Socs3 MBH mice (n 8 or n 7 in each genotype for the vehicle or leptin-treated groups, respectively) sub- jected to an overnight fast was determined by enzyme immunoassay 5 h after injection of vehicle (Veh) or leptin (3.5 mg/kg; i.p.). Data are mean SEM. *p 0.05 and **p 0.01, determined by Bonferroni multiple-comparison test following two-way, repeated-measures ANOVAinAortwo-wayANOVAinB.InA, ##p0.01versusvehicle-treatedSocs3 MBHmice, determined by Bonferroni multiple-comparison test following two-way, repeated-measures ANOVA.

Article Snippet: Membranes were then blocked in 20 mM Tris buffer, pH 7.4, containing 5% bovine serum albumin and 0.1% Tween 20 and incubated with a rabbit polyclonal anti-Socs3 antibody (1:1000; Cell Signaling Technology) overnight at 4°C.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Neuroscience

Article Title: Inactivation of Socs3 in the Hypothalamus Enhances the Hindbrain Response to Endogenous Satiety Signals via Oxytocin Signaling

doi: 10.1523/jneurosci.1669-12.2012

Figure Lengend Snippet: Figure6. Oxytocinrelayshypothalamicactionofleptinontohindbrainsatietycircuits.Schematicrepresentationoftherodent brainshowingtwomajorbrainregionsinvolvedinthecontrolofenergyhomeostasis:thehypothalamus(yellow)andtheNTS(red) in the caudal brainstem. The hypothalamus receives adipose signals such as leptin, and is involved in the long-term regulation of energy balance by fine-tuning various biological processes, including meal size, which is controlled by satiety circuits in the NTS. Here,inactivationofSocs3selectivelyintheMBHofadultmicerevealsacriticalfunctionofSocs3intheregulationofenergybalance in physiological conditions, as this reduced food intake and protected against body weight gain. This Socs3-dependent reduction offoodintakeisexplainedbyanincreasedsensitivitytoendogenous,meal-relatedsatietysignals,mediatedbyoxytocinsignaling inthecaudalbrainstem.WeconcludethatoxytocinlikelyrelayshypothalamicactionofleptinontoNTSsatietycircuits.Thesedata provideananatomicalsubstratefortheeffectofleptinonmealsize,andmoregenerally,amechanismforhowthebraincontrols meal size as a function of the energetic stores available in the organism to maintain energy homeostasis.

Article Snippet: Membranes were then blocked in 20 mM Tris buffer, pH 7.4, containing 5% bovine serum albumin and 0.1% Tween 20 and incubated with a rabbit polyclonal anti-Socs3 antibody (1:1000; Cell Signaling Technology) overnight at 4°C.

Techniques:

Journal: Infection and Immunity

Article Title: Involvement of SOCS3 in Regulation of CD11c + Dendritic Cell-Derived Osteoclastogenesis and Severe Alveolar Bone Loss

doi: 10.1128/iai.01070-08

Figure Lengend Snippet: FIG. 1. Increased expression of SOCS1, SOCS3, and SOCS5 mRNA and proteins is associated with higher alveolar bone loss in vivo. CD4 T cells were purified from cervical lymph nodes of prediabetic, nondiabetic, and diabetic NOD mice with or without OPG treatment (n 5 to 6 mice/group) versus sham-treated (N1 to N3) or IFN- -treated (T1 to T4) HuPBL-NOD/SCID mice (n 10 to 16 mice/group) post-oral infection with A. actinocytmecomitans. qRT-PCR and immunoblot analyses were employed to detect SOCS1, -3, and -5 mRNA and protein versus phosphor-protein expression levels, using ConA-treated CD4 T cells as a positive control. SOCS1, SOCS3, and SOCS5 mRNA and protein expression levels in diabetic, prediabetic, and nondiabetic NOD mice (A) and in IFN- -treated HuPBL-NOD/SCID (T1 to T4) versus sham-treated mice (N1 to N3) (B). The results of qRT-PCR are presented after normalization to -actin expression levels and are representative of 2 to 3 independent studies with similar results. Mean alveolar bone loss in both mouse models was reported previously (27, 54) and is summarized at the bottom of panels A and B.

Article Snippet: Total protein levels were assessed by using antibodies against the mouse and human SOCS1, SOCS3, SOCS5, TRAF6, Erk, JNK1, p38, Akt, and Myc purchased commercially (R&D Systems, Cell Signaling Technology, and Santa Cruz Biotechnology).

Techniques: Expressing, In Vivo, Infection, Quantitative RT-PCR, Western Blot, Positive Control

Journal: Infection and Immunity

Article Title: Involvement of SOCS3 in Regulation of CD11c + Dendritic Cell-Derived Osteoclastogenesis and Severe Alveolar Bone Loss

doi: 10.1128/iai.01070-08

Figure Lengend Snippet: FIG. 2. Increased SOCS3 expression in diabetic CD11c DCs is strongly associated with higher osteoclastogenesis and bone resorption in vitro. A total of 0.5 106 BM-derived immature CD11c DCs prepared from the femurs of diabetic, prediabetic, and nondiabetic NOD mice were cocultured for 4 to 5 days with syngeneic naïve (0.5 106) CD4 T cells, and the sonicated Ag of A. actinomyctemcomitans (Aa). BALB/c total BM cells cultured with M-CSF and RANKL and BM-derived CD11c DCs cultured with A. actinomyctemcomitans were used as positive controls. The cells were then collected on day 4 or 5 and used for the preparation of RNA and cell lysates for subsequent qRT-PCR and Western blot analyses. Representative data from 2 to 3 independent experiments are presented here, with SOCS3 mRNA and protein expression levels (A) and TRAP activity (B) shown from the diabetic, nondiabetic, and prediabetic NOD mice. The results of bone resorption mirrored those of TRAP activity and thus are not shown here.

Article Snippet: Total protein levels were assessed by using antibodies against the mouse and human SOCS1, SOCS3, SOCS5, TRAF6, Erk, JNK1, p38, Akt, and Myc purchased commercially (R&D Systems, Cell Signaling Technology, and Santa Cruz Biotechnology).

Techniques: Expressing, In Vitro, Derivative Assay, Sonication, Cell Culture, Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Infection and Immunity

Article Title: Involvement of SOCS3 in Regulation of CD11c + Dendritic Cell-Derived Osteoclastogenesis and Severe Alveolar Bone Loss

doi: 10.1128/iai.01070-08

Figure Lengend Snippet: FIG. 3. Knocking down functional SOCS3 in CD11c DCs abrogates RANKL-mediated osteoclastogenesis and bone resorption during immune interactions with CD4 T cells in response to A. actinomycetemcomitans sonicated Ags in vitro. Total BM cells of BALB/c, NOD (e.g., diabetic, prediabetic, or nondiabetic) or NOR mice were cultured with IL-4 plus GM-CSF. On day 5, BM cultures were subjected to a transfection protocol with Ad (LacZ) alone or Ad-WT-SOCS3 (or DN-SOCS3) cDNA (DN with F25A mutation with a loss of function) and repeated once again on day 6. On day 7, CD11c DCs were separated by MACS and then cocultured with syngeneic CD4 T cells (1:1 ratio) and A. actinomyctemcomitans for 4 to 5 days before TRAP staining and quantification. Later, cocultured cells were collected and split for the preparation of RNA and cell lysates for subsequent qRT-PCR and immunoblot analyses. Splenocytes plus ConA and total BM plus RANKL plus M-CSF were used as positive controls, while DC-only and DCs plus T cells served as the negative controls. (A) Results after transfection with Ad-DN-SOCS3 or Ad-WT-SOCS3, the results of qRT-PCR for SOCS3 expression levels; (B) immunoblots for SOCS3 protein expression levels. (C and D) The results of TRAP activity analysis. The data for bone resorption mirrored those of TRAP activity and thus are not shown here. The data shown are representative of 2 to 3 independent studies.

Article Snippet: Total protein levels were assessed by using antibodies against the mouse and human SOCS1, SOCS3, SOCS5, TRAF6, Erk, JNK1, p38, Akt, and Myc purchased commercially (R&D Systems, Cell Signaling Technology, and Santa Cruz Biotechnology).

Techniques: Functional Assay, Sonication, In Vitro, Cell Culture, Transfection, Mutagenesis, Staining, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay

Journal: Infection and Immunity

Article Title: Involvement of SOCS3 in Regulation of CD11c + Dendritic Cell-Derived Osteoclastogenesis and Severe Alveolar Bone Loss

doi: 10.1128/iai.01070-08

Figure Lengend Snippet: FIG. 4. Elevated SOCS1, SOCS3, and SOCS5 expression levels are not associated with changes in TRAF6 and MAPK adaptor expression. CD4

Article Snippet: Total protein levels were assessed by using antibodies against the mouse and human SOCS1, SOCS3, SOCS5, TRAF6, Erk, JNK1, p38, Akt, and Myc purchased commercially (R&D Systems, Cell Signaling Technology, and Santa Cruz Biotechnology).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: NOS1-derived nitric oxide promotes NF-κB transcriptional activity through inhibition of suppressor of cytokine signaling-1

doi: 10.1084/jem.20140654

Figure Lengend Snippet: NOS1-derived NO mediates S-nitrosation of SOCS1 and prevents SOCS1-mediated proteasomal degradation of p65 . (A) Immunoblot analysis of p65 and SOCS1 in WT, NOS1 −/− , NOS2 −/− , and NOS3 −/− BMDM treated with LPS (250 ng/ml) for the indicated times. (B) SOCS1 S-nitrosation was detected using the biotin switch method on protein from WT and NOS1 −/− BMDMs pretreated with MG132 (50 µM) for 1 h before treatment with LPS (250 ng/ml) for the indicated times, followed by immunoblotting for SOCS1. (C) NOS2 −/− and NOS3 −/− BMDMs analyzed by biotin switch method, as in B. (D) Co-immunoprecipitation of SOCS1 and p65 in WT and NOS1 −/− BMDMs, pretreated with MG132 (50 µM, 1 h) before LPS (250 ng/ml) for the indicated time intervals. (E) Immunoblot analysis of p65 and SOCS1 total protein levels in NOS1 −/− BMDM treated with DEANO (NO donor, 5 µM) and LPS (250 ng/ml). All blots shown are representative of at least two (C and D) or three (A, B, and E) independent experiments.

Article Snippet: Cells were transiently transfected with pCMV6-AC-GFP Vector (OriGene), which expresses SOCS1 (mouse; available from GenBank under accession no. NM_009896.2 ) driven by the constitutive CMV promoter, using Lipofectamine 2000 reagent according to the manufacturer’s protocol.

Techniques: Derivative Assay, Western Blot, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: NOS1-derived nitric oxide promotes NF-κB transcriptional activity through inhibition of suppressor of cytokine signaling-1

doi: 10.1084/jem.20140654

Figure Lengend Snippet: Molecular modeling and functional testing demonstrate that Cys147 and Cys179 of SOCS1 are the targets for S-nitrosation. (A) Protein domain structure of SOCS1 illustrating the positions of all 5 candidate cysteine residues. (B) Molecular modeling was performed to predict the accessibility of cysteine residues of SOCS1 (represented as colored stick atomic models) to the likely transnitrosation donor, GSNO (represented as ball and stick atomic models). The shorter distances between the cysteine sulfur atom and the nitrogen of GSNO are predicted to permit S-nitrosation for Cys147 (4 Å, middle) and Cys179 (6 Å, right), whereas Cys43 (13 Å, left), Cys112 (11 Å, not depicted), and Cys78 (11 Å, not depicted) are predicted to be too far apart to permit the reaction. (C) Representative immunoblots of GFP-tagged constructs of SOCS1 or point mutations of each cysteine (C147S, C179S, C112S, C78S, and C43S) were transfected into HEK293 cells, which were then treated with a 15-min pulse of 20 ng/ml IL-1β. Some of the cells were pretreated with 50 µM proteasome inhibitor for 1 h (MG132 +) and some were treated with 10 µM DEANO for 1 h after IL-1β (NO donor +). Stability of SOCS1 protein levels was determined by immunoblot and (D) quantified relative to β-actin from three independent experiments (mean ± SEM). (E) The capacity of SOCS1 mutants C147S, and C179S for S-nitrosation was compared with WT SOCS1 in transiently transfected HEK293 cells using the biotin switch method. Immunoprecipitates of GFP-SOCS1 variants were assayed for biotin (S-nitrosation), and lysates were immunoblotted for total SOCS1 as control. Data are representative of two independent experiments.

Article Snippet: Cells were transiently transfected with pCMV6-AC-GFP Vector (OriGene), which expresses SOCS1 (mouse; available from GenBank under accession no. NM_009896.2 ) driven by the constitutive CMV promoter, using Lipofectamine 2000 reagent according to the manufacturer’s protocol.

Techniques: Functional Assay, Western Blot, Construct, Transfection

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: Cavin-1 stability is enhanced in the presence of SOCS3. a HEK293 cells co-transfected with fixed amounts of either myc-tagged FAK1 or GFP-cavin-1 expression constructs and increasing levels of Flag-SOCS3 were treated with or without proteasome inhibitor MG132 (6 μM) as indicated. Detergent-soluble whole-cell lysates were analysed by SDS-PAGE and immunoblotting. b Detergent-soluble whole-cell lysates prepared from SOCS3 +/+ (+/+) and SOCS3 −/− (−/−) MEFs equalised for protein content were analysed by SDS-PAGE and immunoblotting. c Quantitative real-time PCR of cavin-1 mRNA levels in WT ( SOCS3 +/+ ) and SOCS3 −/− MEFs. Data are presented as mean ± standard error for N = 3 experiments. d Upper: Protein-equalised soluble cell extracts from SOCS3 +/+ and SOCS3 −/− MEFs chased for the indicated times in serum-free medium with protein synthesis inhibitor emetine (100 μM) were analysed by SDS-PAGE and immunoblotting with the indicated antibodies. Lower: Quantitation of cavin-1 levels in SOCS3 +/+ and SOCS3 −/− MEFs is also shown. Results are presented as mean values ± standard error for N = 3 experiments. * P < 0.05 vs. corresponding treatment in SOCS3 +/+ MEFs, ψ P < 0.05 vs. t = 0

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Construct, SDS Page, Western Blot, Real-time Polymerase Chain Reaction, Quantitation Assay

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: Effect of SOCS3 deletion on caveola abundance in endothelial cells. a Upper: Detergent-soluble whole-cell lysates from WT and SOCS3-null AS-M.5 human angiosarcoma-derived ECs treated with either vehicle or 50 μM Fsk for 5 h were equalised for protein content for SDS-PAGE for immunoblotting with the indicated antibodies. Lower : Quantitation of cavin-1 and caveolin-1 protein levels in unstimulated AS-M.5 cells is presented as mean ± standard error for N = 3 experiments. * P < 0.05, ** P < 0.01 vs. WT cells. b Transmitting electron microscopy (TEM) was performed on WT and SOCS3-null AS-M.5 cells as indicated. Cell surface caveolae (indicated by the arrows) were readily detectable in WT cells (left panel). In contrast, plasma membranes from SOCS3-null cells were flat and caveolae density was significantly reduced compared to WT cells (right panel). Scale bar = 0.5 μm. c Quantitation of caveola density (number of caveolae per μm of plasma membrane) in WT and SOCS3-null AS-M.5 cells. *** P < 0.0001 vs. WT cells

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Derivative Assay, SDS Page, Western Blot, Quantitation Assay, Electron Microscopy, Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: A region within the SOCS3 SH2 domain is necessary and sufficient for cavin-1 interaction. a Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding Flag-SOCS3 and myc-tagged cavin-1 as indicated were processed by immunoprecipitation (IP) with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. b Protein-equalised soluble cell extracts from WT and SOCS3-null AS-M.5 cells treated with 50 μM Fsk and 6 μM MG132 for 4 h were processed by IP with anti-cavin-1 antibody prior to SDS-PAGE and immunoblotting with the indicated antibodies. As loss of SOCS3 in AS-M.5 cells reduces cavin-1 protein levels ( a ), twice the amount of protein was used as input for SOCS3-null cells to compensate. NS = non-specific band. c Upper: Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either Flag-tagged WT SOCS3 or the indicated truncation mutants and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. Lower: Schematic of the SOCS3 truncation constructs used, KIR = kinase inhibitory region, ESS = extended SH2 subdomain, PEST = PEST (ProGluSerThr) motif. d Upper: Protein-equalised soluble cell extracts from HEK293 cells co-transfected with expression constructs encoding either full-length SOCS3 (WT), a truncated ΔSH2 SOCS3domain (ΔSH2), or SOCS box domain (SB) fused to GFP and myc-tagged cavin-1 as indicated were processed by IP with anti-GFP antibody and protein G-Sepharose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE and for immunoblotting in parallel. Lower: Schematic of the SOCS3-GFP fusions used

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: Cavin-1–SOCS3 interaction occurs independently of the PTyr binding capacity of the SH2 domain. a HEK293 cells transfected with expression constructs encoding Flag-SOCS3 and GFP-tagged cavin-1 as indicated were treated with or without Tyr phosphatase inhibitors sodium orthovanadate (Van: 1 mM) for 1.5 h and then hydrogen peroxide (H 2 O 2 : 0.2 mM) for an additional 30 min prior to harvesting. Protein-equalised soluble cell extracts were then processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. b Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either WT or R71K-mutated Flag-SOCS3 and GFP-tagged-cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. c Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding WT Flag-SOCS3 and GFP-tagged cavin-1 as indicated were incubated with 100 nM N-terminally biotinylated peptides corresponding to the Tyr759 motif of gp130 in its phosphorylated (pY) or non-phosphorylated (Y) forms or a scrambled control (Scr) and streptavidin–agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the pull-down were also fractionated by SDS-PAGE for immunoblotting in parallel

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Transfection, Expressing, Construct, SDS Page, Western Blot, Incubation, Control

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: Cavin-1–SOCS3 interaction requires the SOCS3 SH2 PEST motif. a Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either WT or ΔPEST Flag-SOCS3 were incubated with 100 nM N-terminally biotinylated peptides corresponding to the Tyr759 motif of gp130 in its phosphorylated (pY) or non-phosphorylated (Y) forms and streptavidin–agarose beads prior to loading with whole-cell lysate samples for SDS-PAGE and immunoblotting with the indicated antibodies. b Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either WT or ΔPEST Flag-SOCS3 and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. c Upper, Protein-equalised soluble cell extracts from HEK293 cells transfected with or without the indicated CIS and SOCS3 expression constructs and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. Lower, Schematic of CIS and SOCS3. d Protein-equalised soluble cell extracts from HEK293 cells transfected with or without the indicated SOCS3 and Grap2 expression constructs and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Construct, Incubation, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: SOCS3 interacts with multiple regions within cavin-1. a An immobilised library of 25-mer peptides sequentially shifted by 5 amino acids along the entire cavin-1 open reading frame was overlaid with either purified SOCS3 or a negative control (Cntrl). Dark spots represent areas of interaction between SOCS3 and peptides within the cavin-1 peptide array. The domain structure of murine cavin-1 is indicated below the overlay. b Schematic representation of the N- and C-terminally truncated myc-tagged cavin-1 mutants used for co-IP experiments. c Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either myc-tagged WT cavin-1 or the indicated truncation mutants and Flag-tagged SOCS3 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. *Indicates that expression of the C1 cavin-1 mutant was not detectable; Non Spec refers to immunoglobulin-derived non-specific staining

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Purification, Negative Control, Peptide Microarray, Co-Immunoprecipitation Assay, Transfection, Expressing, Construct, SDS Page, Western Blot, Mutagenesis, Derivative Assay, Staining

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: Cavin-1 drives SOCS3 localisation to the plasma membrane. a WT ( cavin-1 +/+ ) and cavin-1 −/− MEFs transiently expressing SOCS3-GFP (green) were stained with DAPI prior to being fixed, solubilised, and stained with anti-cavin-1 antibody (red) before mounting for imaging by confocal microscopy. Areas of red and green overlap are yellow. Scale bar = 10 μm. b Pearson's correlation coefficient value ( r 2 ) measured from the intensity values located within the rectangular region on the plasma membrane superimposed on the merged cavin-1/SOCS3-GFP image from WT MEFs. c Cavin-1 −/− MEFs transiently co-expressing SOCS3-GFP (green) and cavin-1-mCh (red) were stained with Hoechst 33342 prior to being fixed for imaging by confocal microscopy. Areas of red and green overlap are yellow. Scale bar = 10 μm. d WT, SOCS3 −/− , and cavin-1 −/− MEFs were pre-incubated with Fsk (50 μM) for 5 h to induce SOCS3 prior to subcellular fractionation and analysis by SDS-PAGE and immunoblotting with the indicated antibodies

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Clinical Proteomics, Membrane, Expressing, Staining, Imaging, Confocal Microscopy, Incubation, Fractionation, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: Cavin-1 limits Tyr705 phosphorylation of STAT3 via SOCS3. a Upper: Protein-equalised soluble cell extracts from cavin-1 +/+ and cavin-1 −/− MEFs treated for the indicated times with sIL-6Rα/IL-6 (25 and 5 ng ml −1 ) were fractionated by SDS-PAGE prior to immunoblotting with the indicated antibodies. Quantitation of normalised Tyr705 phospho-STAT3 in cavin-1 +/+ and cavin-1 −/− MEFs are presented as mean values ± standard error for N = 3 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding treatment in cavin-1 +/+ MEFs. b Protein-equalised soluble cell extracts from cavin-1 +/+ and cavin-1 −/− MEFs treated for 30 and 60 min with leukaemia inhibitory factor (LIF; 0.5 nM), oncostatin M (OSM; 10 ng ml −1 ) or vehicle (V) were fractionated by SDS-PAGE prior to immunoblotting with the indicated antibodies. c Upper: WT ( cavin-1 +/+ ) and cavin-1 −/− MEFs were pre-incubated with or without Fsk (50 μM) for 5 h prior to treatment with or without sIL-Rα/IL-6 (25 and 5 ng ml −1 ) for 30 min. Cell extracts were analysed by SDS-PAGE and immunoblotting with the indicated antibodies. Lower: Quantitation of normalised Tyr705 phospho-STAT3 and SOCS3 in cavin-1 +/+ and cavin-1 −/− MEFs is presented as mean ± standard error for N = 3 experiments, * P < 0.05 vs. sIL-6Rα/IL-6 treatment in cavin-1 +/+ MEFs, # P < 0.05 vs. vehicle treatment in cavin-1 −/− MEFs.

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, SDS Page, Western Blot, Quantitation Assay, Incubation

Journal: Nature Communications

Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

doi: 10.1038/s41467-017-02585-y

Figure Lengend Snippet: Model of novel functional interactions between SOCS3 and cavin-1

Article Snippet: Human SOCS3 CRISPR/Cas9 knockout (KO) and human SOCS3 HDR plasmids (cat. no. sc-400455) were from Santa Cruz Biotechnology.

Techniques: Functional Assay