Journal: Oncotarget

Article Title: SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma

doi: 10.18632/oncotarget.7923

Figure Lengend Snippet: A. Left , The transient effect of SHP-1 on p-PKM2 Y105 . PLC5 and Hep3B cells were transiently transfected with PKM2 for 48 h. Right , SHP-1 induces PKM2 Y105 dephosphorylation in HCC cells stably expressing SHP-1. B. Silencing SHP-1( PTPN6 ) upregulates p-PKM2 Y105 . C. Identification of PKM2 as an interaction partner of SHP-1. The HCC cell lysate (1 mg) were used in immunoprecipitation. D. Silencing SHP-1 did not further affect the phospho-status of PKM2 in cells transfected with PKM2 Y105F mutant. PLC5 cells transfected with wild-type or mutant PKM2 and SHP-1( PTPN6 ) siRNA or control siRNA were analyzed by IP and IB as indicated. E. PKM2 is a phospho-substrate of SHP-1 as demonstrated by substrate trapping assay. The interaction affinity of wild-type or catalytic-dead SHP-1 (C453S) with PKM2 was assayed.

Article Snippet: Plasmid for pJ3-SHP-1 (C453S) was purchased from Addgene plasmid repository ( http://www.addgene.org/ ).

Techniques: Transfection, De-Phosphorylation Assay, Stable Transfection, Expressing, Immunoprecipitation, Mutagenesis, Control

Journal: International Journal of Molecular Sciences

Article Title: Tamoxifen Exerts Anticancer Effects on Pituitary Adenoma Progression via Inducing Cell Apoptosis and Inhibiting Cell Migration

doi: 10.3390/ijms23052664

Figure Lengend Snippet: TAM activated TLR4 signaling and inactivated immune checkpoint SHP1/SHP2. ( A ) The protein expression levels of the TLR4/NF-κB signaling pathway normalized to the GAPDH in RAW 264.7 cells were detected. ( B ) The expression levels of SIRPα protein, the phosphorylation levels of SHP1 and SHP2 proteins in RAW 264.7 cells were detected using Western blot. ( C , D ) The expression levels of SIRPα protein, the phosphorylation levels of SHP1 and SHP2 proteins in GH3 and AtT-20 cells were detected. ( E ) The expression levels of SIRPα protein, the activity of SHP1 and SHP2 proteins in tumor tissues were also detected. Bars indicates SEM, VC vehicle control, refers to tumor-bearing mice treated with the vehicle. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control or VC. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. cells treated with IL-TAM: tamoxifen.

Article Snippet: Western blotting was performed as per standard protocols, and the membranes were probed with antibodies targeting p-JAK1 (74129, Rt, 1:500), JAK1 (29261, Rt, 1:1000), p-STAT6 (56554, Rt, 1:500), p-PI3K (17366, Rt, 1:500), PI3K (4249, Rt, 1:1000), p-AKT (4060, Rt, 1:500), AKT (4685, Rt, 1:1000), p53 (48818, Ms, 1:1000), Bax (14796, Rt, 1:1000), p-NF-κB p65 (3033, Rt, 1:500), NF-κB p65 (8242, Rt, 1:1000), p-IκBα (2859, Rt, 1:500), IκBα (4814, Ms, 1:1000), p-IKKα/β (2697, Rt, 1:500), IKKα (11930, Ms, 1:1000), IKKβ (8943, Rt, 1:1000), p-SHP1 (8849, Rt, 1:500), SHP1 (26516, Rt, 1:1000), p-SHP2 (5431, Rt, 1:500), SHP2 (3397, Rt, 1:1000), SIRPα (47027, Rt, 1:1000) (Cell Signaling, Danvers, MA, USA), STAT6 (YT4454, Rt, 1:1000), MyD88 (YM33092, Ms, 1:1000) (Immunway, Plano, MA, USA), GAPDH (10494-1-AP, Ms, 1:1000), INOS (22226-1-AP, Rt, 1:1000) (Proteintech, Wuhan, Hubei, China), Bcl-2 (ab32124, Rt, 1:1000) (Abcam, Cambridge, MA, USA) and TLR4 (sc-293072, Ms, 1:1000) (santa cruz biotechnology, Silicon Valley, CA, USA)positive bands were detected using an ECL Kit (CWBIO, Beijing, China) and the Tanon Chemiluminescence Image Analysis System (Shanghai, China).

Techniques: Expressing, Phospho-proteomics, Western Blot, Activity Assay, Control

Journal: Frontiers in immunology

Article Title: SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients.

doi: 10.3389/fimmu.2020.610523

Figure Lengend Snippet: FIGURE 5 | SHP1 but not SHP2 is downstream of SIRPa. (A) Total and phosphorylated SHP1 and SHP2 expression were examined in phenotypically sensitive and resistant NLCs by western blotting. Representative blots and quantified data are shown. Each point represents a single patient. (B) NLCs were transfected with SIRPa siRNA C or scrambled control siRNA. SIRPa protein expression, as well as (C) total and phosphorylated SHP1 (pSHP1) or SHP2 (pSHP2) expression was estimated in transfected NLCs. Representative blots are shown and quantified data shown graphically. Data presented as mean value ± SEM and normalized to scrambled siRNA control. Each point represents an individual patient. Statistical differences were calculated by a Mann Whitney U Test. ***p < 001 and ****p < 0.0001. ns, not significant.

Article Snippet: Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or b-actin or GAPDH using Image J software. siRNA Knockdown Predesigned stealth small interfering RNAs (siRNAs) were used to silence protein expression of SIRPa (27mer Human siRNA duplexes; SR315411; OriGene) and Shp1 (27mer Human siRNA duplexes; SR321517; OriGene).

Techniques: Expressing, Western Blot, Transfection, Control, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients.

doi: 10.3389/fimmu.2020.610523

Figure Lengend Snippet: FIGURE 7 | SHP1-dependent ADP repression is SYK-dependent. NLCs were transfected with 50 nM scrambled siRNA control or three different SHP1-siRNAs (siRNA A–C). (A) Protein was harvested after 48 h and SHP1 and GAPDH expression visualized and quantitated by western blot to examine knockdown efficiency. (B) NLCs were co- cultured with autologous CLL cells for 48 h after SHP1 siRNA C or scrambled siRNA transfection. Cultures were then treated with or without obinutuzumab (10 µg/ml) for 2 h before ADP responses examined. Data presented as mean value ± SEM from five CLL patients. (C) Western blot data analysis of SYK (pSYKY525/526) and AKT phosphorylation (pAKTS473) in NLCs following SHP1 siRNA transfection. Representative blots and quantified data are shown. Data presented as mean value +/−SEM. Each point represents an individual patient. Statistical analysis used a Mann Whitney U Test. *p < 0.05, ***p, 0.001, and ****p < 0.0001.

Article Snippet: Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or b-actin or GAPDH using Image J software. siRNA Knockdown Predesigned stealth small interfering RNAs (siRNAs) were used to silence protein expression of SIRPa (27mer Human siRNA duplexes; SR315411; OriGene) and Shp1 (27mer Human siRNA duplexes; SR321517; OriGene).

Techniques: Transfection, Control, Expressing, Western Blot, Knockdown, Cell Culture, Phospho-proteomics, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients.

doi: 10.3389/fimmu.2020.610523

Figure Lengend Snippet: FIGURE 8 | Overview of FcgR signaling pathway in antibody resistant NLC. Therapeutic antibody binding to cell-surface receptor, FcgR independently activates SYK/BTK and p110d/AKT pathways. Activation of SIRPa by tumor cell CD47 (not shown) activates SHP1 phosphatase that suppresses SYK activity.

Article Snippet: Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or b-actin or GAPDH using Image J software. siRNA Knockdown Predesigned stealth small interfering RNAs (siRNAs) were used to silence protein expression of SIRPa (27mer Human siRNA duplexes; SR315411; OriGene) and Shp1 (27mer Human siRNA duplexes; SR321517; OriGene).

Techniques: Binding Assay, Cell Surface Receptor Assay, Activation Assay, Activity Assay

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) expression is downregulated in dilated cardiomyopathy (DCM) human hearts and aortic banding (AB)-operated mouse hearts. A and B, Protein levels of β-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), and SIRPA in samples from donor hearts and DCM hearts (n=3; *P<0.05 vs donor hearts). C and D, Protein levels of β-MHC, ANP, and SIRPA in samples from wild-type mice at the indicated times after sham or AB surgery (n=3; *P<0.05 vs sham). E and F, Protein levels of β-MHC, ANP, and SIRPA in samples from neonatal rat cardiomyocytes treated with angiotensin II (Ang II; 1 μmol/L) or phenylephrine (PE; 100 μmol/L) for 48 h (n=3; *P<0.05 vs PBS). Representative blots (A, C, and E) and quantitative results (B, D, and F). n indicates number of independent experiments.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Expressing

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) deficiency augments pressure overload–induced cardiac hypertrophy and fibrosis. A, Images of heart sections stained with H&E and WGA from wild-type (WT) and knockout mice 4 weeks after sham or aortic banding (AB) surgery (n=6–10 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=13–15 mice per experimental group). F, Images of heart sections stained with picrosirius red from WT and knockout mice 4 weeks after sham or AB surgery (n=6–10 mice per experimental group). G, Statistical results for the left ventricular (LV) collagen volume (%; n=>40 fields). *P<0.05 vs WT/sham; #P<0.05 vs WT/AB.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Staining, Knock-Out

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α overexpression mitigates aortic banding (AB)-induced cardiac remodeling. A, Images of heart sections stained with H&E and WGA from nontransgenic (NTG) and transgenic (TG) mice 8 weeks after sham or AB surgery (n=6 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=14–15 mice per experimental group). F, Images of heart sections stained with PSR from NTG and TG mice 8 weeks after sham or AB surgery (n=6 mice per experimental group). G, Statistical results for the left ventricular collagen volume (%; n=>40 fields). *P<0.05 vs NTG/sham; #P<0.05 vs NTG/AB.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Over Expression, Staining, Transgenic Assay

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) inhibits the Toll-like receptor 4 (TLR4) signaling pathway. A and B, Protein expression levels of TLR4 are upregulated in the SIRPA knockout mice but downregulated in the SIRPA transgenic mice 2 weeks after aortic banding (AB) surgery (n=3; *P<0.05 vs wild-type [WT] or nontransgenic (NTG)/sham; #P<0.05 vs WT or NTG/AB). Representative blots (top); quantitative results (bottom). C, Representative colocalization images of SIRPA and TLR4 in HEK293T cells. Red, TLR4; green, SIRPA; blue, nucleus. D, SIRPA interacts with TLR4. Western blot with the Flag or Myc antibody after the coimmunoprecipitation of TLR4 from HEK293T whole-cell lysates using the Flag antibody (left). Western blot with the Flag or Myc antibody after the coimmunoprecipitation of SIRPA from HEK293T whole-cell lysates using the Myc antibody (right). E and F, Phosphorylation and total protein levels of IκBα and P65 in samples from (E) WT and SIRPA knockout mice and (F) NTG and SIRPA transgenic mice 2 weeks after AB surgery (n=3; *P<0.05 vs WT or NTG/AB). Representative blots (top); quantitative results (bottom). n indicates number of independent experiments.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Expressing, Knock-Out, Transgenic Assay, Western Blot, Phospho-proteomics

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Disruption of Toll-like receptor 4 (TLR4) rescued the adverse effect of signal regulatory protein-α (SIRPA) deficiency on cardiac hypertrophy. A, Images of heart sections stained with H&E and WGA from the indicated groups (n=5 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=11 mice per experimental group). F and G, Phosphorylation and total protein levels of IκBα and P65 in samples from the indicated groups 4 weeks after aortic banding(AB) surgery (n=3 independent experiments). Representative blots (F); quantitative results (G). DKO indicates double knockout.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Disruption, Staining, Phospho-proteomics, Double Knockout